Development of a marker foot and mouth disease virus vaccine candidate that is attenuated in the natural host

ABSTRACT

We have generated novel molecularly marked FMDV A 24 LL3D YR  and A 24 LL3B PVKV 3D YR  vaccine candidates. The mutant viruses contain a deletion of the leader coding region (LL) rendering the virus attenuated in vivo and negative antigenic markers introduced in one or both of the viral non-structural 3D pol  and 3B proteins. The vaccine platform includes unique restriction endonuclease sites for easy swapping of capsid proteins for different FMDV subtypes and serotypes. The mutant viruses produced no signs of FMD and no shedding of virulent virus in cattle. No clinical signs of disease or fever were observed and no transmission to in-contact animals was detected in pigs inoculated with live A 24 LL3D YR . Cattle immunized with chemically inactivated vaccine candidates showed an efficacy comparable to a polyvalent commercial FMDV vaccine. These vaccine candidates used in conjunction with a cELISA provide a suitable target for DIVA companion tests.

This application is a divisional application of application Ser. No.13/157,097 filed Jun. 9, 2011, now U.S. Pat. No. 8,765,141, which claimsthe benefit of U.S. Provisional Application No. 61/360,719 filed Jul. 1,2010, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a rationally designed engineered attenuatedantigenic marker vaccine-virus production platform comprising a deletionof the L^(pro) coding sequence resulting in complete attenuation andmutations (negative markers) introduced in two non-structural viralproteins resulting in the elimination of two antigenic epitopesrecognized by specific antibodies, one located in 3B and the other in3D, thus providing a target for DIVA (Differentiation of naturallyInfected from Vaccinated Animals) serological tests. The attenuatedmarker vaccine production virus also comprises unique restrictionendonuclease sites flanking the capsid-coding region to facilitate thereplacement of the capsid region making possible the exchange ofcassettes representing relevant capsid coding regions of differentserotypes and subtypes of FMDV field isolates for the design of customvaccines.

2. Description of the Relevant Art

Foot and mouth disease (FMD) is an extremely contagious viral disease ofcloven-hoofed ungulates which include domestic animals (cattle, pigs,sheep, goats, and others) and a variety of wild animals. The mostprominent disease symptoms in FMDV-infected cattle include vesicularlesions of the epithelium of the mouth, tongue, teats and feet. Althoughsome countries, among them United States, Canada, Mexico, Australia andmost of Europe, are considered to be free of FMD, the disease isdistributed worldwide and has a great economic impact on the exportindustry. Indeed, several economically devastating outbreaks haveoccurred over the past decade on almost every continent.

Control methods to eradicate FMD depend upon the prevalence of thedisease in particular geographical regions/states and often include massannual prophylactic vaccination campaigns and the application ofstringent zooprophylactic measures following outbreaks. A chemicallyinactivated whole virus vaccine has been used to contain the disease,but it is slow acting and does not always permit distinction betweeninfected and vaccinated animals. Indeed, in recent years thedifferentiation of infected animals from those that have been vaccinatedis of paramount importance as a protective activity following emergencyvaccination. Historically, the use of non-structural viral protein asserological indicators of viral replication has been widely applied.Among these proteins, the highly conserved FMDV 3D polymerase (3D^(pol))of 52-KDa has been identified as the main determinant of infection andhas been called the FMD-Virus Infection-Associated Antigen (FMD-VIAA;Berger et al. 1990. Vaccine 8:213-216; Bergmann et al. 1993. Am. J. Vet.Res. 54:825-831; Cowan and Graves. 1966. Virology 30:528-540; McVicarand Sutmoller. 1970. Am. J. Epidemiol. 92:273-278; Sorensen et al. 1998.Arch. Virol. 143:1461-1476). Studies by Newman and Brown (1997. J.Virol. 71: 7657-7662; Newman et al. 1994. Proc. Natl. Acad. Sci. USA91:733-737) suggested that purified 140S FMDV preparations contain smallquantities of 3D^(pol) and therefore, could account for seroconversionto 3D^(pol) in animals that have received inactivated FMDV vaccines.

Previous strategies to select highly attenuated vaccines for FMDV haverelied on the selection of less-pathogenic variants produced by serialpassages of the virus in non-natural hosts such as embryonated chickeneggs and rabbits (Giraudo et al. 1990. Virology 177:780-783; Xin et al.2009. Vet. Microbiol.). Those empirical strategies failed when tested insusceptible species due to reversion to virulence by the mutant virusesharboring point-mutations and therefore, were not pursued for being toorisky (Sutmoller, P. 2001. Rev. Sci. Tech. 20:715-722; Sutmoller et al.2003. Virus Res. 91:101-144). Modern approaches to produce geneticallyengineered FMDV with altered virulence have relied on the deletion ofthe cell-receptor binding site (Mason et al. 1994. Proc. Natl. Acad.Sci. USA 91:1932-1936; McKenna et al. 1995. J. Virol. 69:5787-5790;Rieder et al. 1996. Proc. Natl. Acad. Sci. USA 93:10428-33), the viralleader coding sequence (L^(pro), Piccone et al. 1995. J. Virol.69:5376-82) or elements within the non-translated region (NTR)(Rodriguez et al. 2009. J. Virol. 83:3475-3485). The FMDV L^(pro)together with the 3C^(pro) and 2A proteinases play an important role inprocessing of the viral polyprotein. In addition to cleaving itself fromthe nascent polyprotein, L^(pro) cleaves the eukaryotic initiationfactor 4G (eIF4G) causing inhibition of the cellular translationmachinery. L^(pro) is also known to relocate to the nucleus in theFMDV-infected cells and to induce degradation of nuclear factor kappa B(NF-κB) with the consequent inhibition of the innate immune response (deLos Santos et al. 2006. J. Virol. 80:1906-1914). FMDVs of serotype Alacking L^(pro) have been shown to be infectious, to grow more slowly inBHK-21 cells (A₁₂-LLV2, (Piccone et al., supra) and be attenuated forpigs (Brown et al. 1996. J. Virol. 70:5638-5641; Chinsangaram et al.1998. Vaccine 16:1516-1522; Mason et al. 1997. Virology 227:96-102).

Currently killed-antigen FMDV vaccines are produced in expensivebiological containment facilities, by growing large volumes (thousandsof liters) of virulent FMDV that has been adapted to grow in cells,which can be sometimes difficult. This process has resulted in escape ofvirulent virus from the manufacturing facility causing costly outbreaksin livestock (see Cottam et al. 2008. PLoS Pathogen 4:1-8). Aftergrowth, virus is then inactivated using chemicals and antigenconcentrates are prepared, followed by purification steps required toremove contaminant proteins, making it difficult to differentiateinfected from vaccinated animals (DIVA) through serological diagnostictests. There is little to no cross protection across serotypes andsubtypes requiring the appropriate matching between vaccine andcirculating field strains to achieve protection. Despite theseshortcomings of the vaccines, billions of doses are manufactured everyyear around the world. Their use has been the basis for eradicating FMDVfrom Europe and for controlling the disease in many parts of the worldthrough mass vaccination campaigns. Thus, there is an urgent need forthe development of effective marker FMDV vaccine candidates with DIVAcapabilities.

SUMMARY OF THE INVENTION

We have discovered a novel, safe, molecular-based attenuated FMD virusvaccine platform for FMD control and eradication; the vaccine hasnegative markers that allows the differentiation of naturally infectedanimals from vaccinated animals.

In accordance with this discovery, it is an object of the invention toprovide a recombinant viral-vectored vaccine platform comprising DNAencoding a genetically modified FMDV vector that is attenuated in thenatural host by design comprising mutations (negative markers)introduced in two non-structural viral proteins resulting in theelimination of two antigenic epitopes recognized by specific antibodies,one located in protein 3B and the other in protein 3D, thus providingtwo possible targets for DIVA (Differentiation of naturally Infectedfrom Vaccinated Animals) serological tests. The vaccine platform alsocomprises unique restriction endonuclease sites to facilitate thereplacement of the capsid region making possible the exchange ofcassettes representing relevant capsid coding regions of differentserotypes and subtypes of FMDV field isolates.

It is thus an object of the invention to provide an isolatedpolynucleotide molecule comprising a genetically modified DNA sequenceencoding a genetically modified FMDV. The FMDV is genetically modified,i.e., it is a leaderless virus containing a deletion of the leader(L^(pro)) protein coding region such that FMD viruses lacking thisprotein are attenuated in cattle and pigs.

It is additionally an object of the invention to provide a geneticallymodified FMDV encoded by the isolated polynucleotide molecule recitedabove and further containing an alteration in the sequence of one ormore of the non-structural viral proteins where there is an insertion ofa conserved B cell immunodominant epitope in a virus non-structuralprotein(s) providing a negative marker vaccine that is attenuated in thenatural host by design and that can elicit an immune response that canbe distinguished from the immune response induced by the field virus.

It is a further object of the invention to provide a geneticallymodified FMDV encoded by the isolated polynucleotide molecule recitedabove where the alteration is in the sequence of one of thenon-structural viral proteins, 3D^(pol), and where the alteration is asubstitution at position H27Y and N31R, resulting in the geneticallymodified FMDV LL[YR].

An added object of the invention is to provide a genetically modifiedFMDV encoded by the isolated polynucleotide molecule recited above wherethe alteration is in the sequence of one of the non-structural viralproteins, 3D^(pol), and also a mutation in 3B (RQKP→PVKV, found inBRV-2) that abolishes reactivity with MAb F8B.

Another object of the invention is to provide a marker FMDV cDNA clonethat is further modified for inclusion of unique restrictionendonuclease sites to facilitate the replacement of the capsid region,thus making possible a cassette design allowing for rapid replacement ofparental capsid sequences with donor capsid sequences from differentFMDV subtypes and serotypes.

An additional object of the invention is to provide a recombinantviral-vectored vaccine platform for production of chemically-inactivatedFMD vaccine comprising a genetically modified FMDV comprising deletionof the L^(pro) coding sequence, a mutation in a B cell immunodominantepitope in the virus non-structural protein 3D^(pol) or mutations in Bcell immunodominant epitopes of both 3D^(pol) and 3B viralnon-structural proteins, inclusion of unique restriction endonucleasesites to facilitate the replacement of the capsid region of new viralstrains.

Another object of the invention is to provide a rationally designedattenuated FMDV vaccine that used in a chemically-inactivated form iseffective to protect an animal from clinical FMD when challenged withvirulent FMDV wherein said vaccine comprises a FMD leaderless virushaving unique restriction endonuclease sites to facilitate thereplacement of the capsid region.

A further object of the invention is to provide a marker vaccine whichallows a serological distinction between vaccinated animals and animalsinfected with FMDV.

A still further object of the invention is to provide a strategy formaking a FMDV-vectored vaccine platform, which method comprises agenetically engineered attenuated FMDV backbone, molecularly marked byinsertion of one or more conserved B cell immunodominant epitopes from avirus different from, but related to, FMDV, and further modified by theinclusion of unique restriction endonuclease sites.

Yet another object of the invention is to provide a method forprotecting an animal against FMD by administering an effective amount ofrationally designed and chemically-inactivated marker FMDV vaccine.

An additional object of the invention is to provide a method fordelaying onset or severity of FMD in an animal by administering aneffective amount of rationally designed and chemically-inactivatedmarker FMDV vaccine.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the U.S. Patent and TrademarkOffice upon request and payment of the necessary fee.

FIG. 1 depicts the polyacrylamide gel electrophoresis (PAGE) of theradioimmunoprecipitation reactions of ³⁵[S] methionine-labeled FMDV orBovine Rhinovirus type 2 (BRV2) viral proteins with rabbit antisera ormonoclonal antibodies (MAbs) F32-44 or F19-59 raised againstFMDV-3D^(pol). The 3D protein was separated by 12% SDS polyacrylamidegel electrophoresis. The gel was fixed, dried, and exposed to X-rayfilm.

FIG. 2A depicts a schematic representation of the FMDV genome andrelative locations of the modifications introduced in the viruses usedin this study. A₂₄LL3_(DYR) virus was generated by site directedmutagenesis of a full-length clone pA₂₄Cru of the FMDV outbreak strainA₂₄ Cruzeiro. Additional modifications introduced in the mutant plasmidincluded: δL: deletion of the leader gene; underlined are two amino acidsubstitutions at position H₂₇Y and N₃₁R of the FMDV 3D^(pol): A₂₄LL (SEQID NO:5), A₂₄LL3D_(YR) (SEQ ID NO:6); two unique restrictionendonuclease enzyme cloning sites 1 and 2 (♦, RE1, RE2); Deletedantigenic determinant in the genome (3DYR; ▴). FIG. 2B depicts theradioimmuno-precipitation of ³⁵S-radiolabled viral 3D^(pol) protein withselected MAbs. Extracts were prepared from cell lysates obtained fromBHK-21 cells infected or not with A24WT, A24LL or A24LL3DYR viruses at aMOI of 5 PFU/cell. The cell extracts were run under denaturingconditions in a 12% SDS-PAGE gel as described in Example 4. Thenitrocellulose blots were probed with MAb F19-59 and F32-44 for FMDV3D^(pol) protein.

FIG. 3A shows 24 h single growth curves of FMDVs. Cell monolayers weremock- or infected with A₂₄WT, A₂₄WT3D_(YR), A₂₄LL or A₂₄LL3D_(YR)viruses at a MOI of 5 PFU/cells. Procedures used for viral infection andtitration of infectivity are described in Example 3. Each valuerepresents the mean of triplicate assays. FIG. 3B shows the plaquephenotypes of A₂₄WT and mutant FMDVs on BHK-21. BHK-α_(V)β₆ or LFBK cellmonolayers are also shown. FIG. 3C depicts the analysis of theexpression of foreign epitope by mutant FMDVs. BHK-21 cells wereinfected with the parent recombinant virus A₂₄WT, A₂₄WT3D_(YR) orA₂₄LL3D_(YR). At 8 hpi, cells were fixed and processed for IHC usingMAbs specific for 3D^(pol) protein (F19-59, F32-44). As expected, MAbF32-44 reacted only with WT 3D^(pol) protein, and the F19-59 MAbrecognized an epitope contained on both virus proteins.

FIG. 4 shows the results of a Competitive Enzyme-Linked ImmunoabsorbentAssay (cELISA) measuring the differential antibody response in animalsinfected with A₂₄WT and A₂₄WT3D_(YR) using MAb F32-44 (Accession No.130514-02, International Depositary Authority of Canada, Winnipeg,Manitoba, Canada) which specifically binds to an epitope of 3D^(pol).Each group shows the average ±1 SD of 2-3 cows infected with eithervirus. Samples were collected before inoculation and at necropsy. Normalbovine serum (NBS) was used as negative control (no inhibition).^(#)DPI: days post inoculation. * IDL: intradermolingual.

FIG. 5 is a schematic representation of the FMDV genome and features ofthe marker FMDV vaccine platform.

FIG. 6A is a schematic representation of the double negative marker FMDviruses (top panel). The FMDV MAb F8B epitope in 3B (SEQ ID NO:15) andMAb F32-44-epitope in 3D^(pol) (SEQ ID NO:5) are shown along with theirmodified versions, 3B_(PVKV) (SEQ ID NO:16) and 3D_(YR) (SEQ ID NO:6),respectively, where the MAb reactivities were abolished in the mutantviruses. FIG. 6B depicts the antigenic phenotype of marker FMDVscompared to the parental virus using Western blot analyses. FIG. 6Cshows plaque phenotypes of the double negative antigen mutant viruses inthe wild-type and LL backbones in comparison to the WT virus.

FIG. 7 shows the results of a Competitive ELISA (cELISA) measuring thedifferential antibody response in animals infected withA₂₄WT3B_(PVKV)3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) viruses using MAb F8B(Accession No. 130514-01, International Depositary Authority of Canada,Winnipeg, Manitoba, Canada) which specifically binds to an epitope of3B. Each graph shows the results of individual cow sera collected atdays 0 (before inoculation, no inhibition) and 21 dpi. ^(#)DPI: dayspost inoculation.

FIG. 8A is a schematic representation of the wild type and chimera FMDVgenomes and relative locations of the modifications introduced in theviruses used in this study. A₂₄LL Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) construct were generated by exchangingthe capsid fragments from FMDV Asia1 Shamir and FMDV Turkey06,respectively and inserting into the p A₂₄LL3B_(PVKV)3D_(YR) infectiousclone derived from the FMDV outbreak strain A₂₄ Cruzeiro. Additionalmodifications present in the mutant plasmids included: ΔL: deletion ofthe leader gene; 3B₂₃: only two copies of 3B genes; 3D^(pol): two aminoacid substitutions at position H₂₇Y and N₃₁R; two unique restrictionendonuclease enzyme cloning sites 1 and 2 (♦, RE1, RE2). Titrations ofinfectivity for A₂₄WT and chimeric FMDVs viruses were performed asstated in Example 6. The plaque phenotypes on BHK-21, LFBK, IBRS2 cellmonolayers are shown. FIG. 8B depicts a diagnostic assay for detectionof chimera FMDV viruses. A₂₄WT, A₂₄LL Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) viruses were passed in BHK-21 cellsfour times and viral RNAs were extracted. RT-PCR reactions wereperformed as outlined in Example 6 using primers to detect presenceand/or absence of specific mutations in chimeric viruses.

FIG. 9 shows 48 h single growth curves of A₂₄WT and chimera FMDVs. Cellmonolayers were mock- or infected with A₂₄WT, A₂₄LLAsia1-A₂₄LL3B_(PVKV)3D_(YR) and A/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR)viruses at a MOI of 5 PFU/cells. Procedures used for viral infection aredescribed in Example 6.

FIG. 10 depicts the analysis of the expression of foreign epitopes bychimera FMDVs. BHK-21 cells were mock- or infected with the parentrecombinant virus A₂₄WT, A₂₄LL Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) at a MOI of 5 PFU/cells. At 5 hpi,cell lysates were collected and run under denaturing conditions in a 12%SDS-PAGE gel as described in Example 6. The nitrocellulose blots wereprobed with MAbs F8B for FMDV 3B protein and F19-6 and F32-44 for FMDV3D^(pol) protein.

DETAILED DESCRIPTION OF THE INVENTION

To enable the implementation and successful outcome of FMD control anderadication campaigns, new vaccines should ideally possess a productprofile with several key attributes: (1) confers rapid (<7 days)protection against generalized disease following single doseimmunization, (2) prevents viral shed, disease transmission and carrierstate following direct contact exposure, (3) provides at least 12 monthsprotective immunity following a 2-dose regimen, (4) maintainsthermostablity for at least 3 months in the cold liquid state, (5)provides broad intra-serotype protection, (6) provides a manufacturingprocess that eliminates the need for high containment facilities, and(7) contains a negative marker that is DIVA compatible.

This study describes the experimental development of a marker FMDVvaccine production platform candidate, which creates new potentialitiesfor the control of FMD. The negative antigenic marker virusesA₂₄LL3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) derived by infectious cDNAtechnology, lack the region coding for L^(pro) and contains areplacement of an immunodominant epitope in 3B and 3D^(pol) by thecorresponding sequence of bovine rhinovirus that serves as negativeantigenic epitope in these proteins. Aerosol inoculation of the livevirus in cattle show limited growth and absence of FMD signs in theinoculated animals and no transmission to naïve animals in directcontact. Moreover, the animals did not shed significant amount of virusto the environment. Likewise, swine inoculated in the heal-bulb withA₂₄LL3D_(YR) virus, showed no clinical signs of FMD. Furthermore, theinoculated animal did not transmit the disease to contact animals. TheA₂₄LL3D_(YR) vaccine candidate used to produce inactivated antigen bychemical binary ethylenimine (BEI) inactivation proved to be aseffective as a commercially available inactivated antigen FMDV vaccinein protecting cattle and swine from challenge with the parental FMDvirus.

Picornaviruses contain a positive sense single-strand RNA genome thatencodes a single polyprotein that is processed to produce bothnon-structural (NSP, replicating) and structural (SP, capsid) proteins.In FMD control and livestock surveillance programs, the use of expressedrecombinant NSP products (baculovirus, E. coli) coupled with diagnosticassays such as ELISA (competitive, indirect) enzyme-linkedimmuno-electrotransfer blot (EITB), VIAA have been extensively exploitedto allow discrimination between animals which have been vaccinatedagainst FMD from those that have recovered from infection (Fernándes etal. 1990. Prev. Vet. Med. 9:233-240; Bergmann et al. 2000. Arch. Virol.145:473-489; Brocchi et al. 2006. Vaccine 24:6966-6979; Clavijo et al.2004. J. Virol. Methods 120:217-227; Dekker et al. 2008. Vaccine26:2723-2732; McVicar and Sutmoller, supra; Sorensen et al. 2005. Arch.Virol. 150:805-814; Sorensen et al. 1998, supra; Yang et al. 2007b. Vet.Immunol. Immunopathol. 115:126-134). Vaccinated animals that are exposedto virus might be infected without clinical manifestations of FMD andsubsequently become chronic carriers representing potential sources fornew outbreaks of the disease. Therefore, there is a need to develop moreeffective vaccines that block virus infection and that do not induceantibodies against some of the immunogenic non-structural viral proteinsproduced during FMDV replication in the host in order to differentiatethe response of naturally infected animals from vaccinated animals.

In this study we present an approach to rationally design novel negativemarker FMDV vaccine viruses. The vaccine candidate viruses A₂₄LL3D_(YR)and A₂₄LL3B_(PVKV)3D_(YR) harbor negative antigenic markers forpotential DIVA capabilities, encoded in either the 3D^(pol) alone or 3Band 3D^(pol) combined, respectively. Additional modification of thevaccine virus consisted of the deletion of the non-essential L^(pro)coding sequence that rendered these viruses attenuated in vivo.Therefore, it could reduce the risk for escape of virulent FMDV duringlarge scale during vaccine production. The vaccine platform alsoincludes strategically-located restriction-enzyme sites that allow easyswapping of the relevant antigenic region for different serotypes andsubtypes.

Comparison of the 3D^(pol) sequence for FMDV and BRV-2 viruses revealed64% identity (Hollister et al. 2008. Virology 373:411-425) at the aminoacid level. Amino acids 16-32 comprise an important antigenic site inthe FMDV 3D^(pol) protein (Yang et al. 2007a. J. Immunol. Methods321:174-181) and this peptide is 76% identical among theseclosely-related viruses. MAb F32-44 (Accession No. 130514-02, depositedMay 13, 2014, International Depositary Authority of Canada, 1015Arlington Street, Winnipeg, Manitoba, Canada R3C 3R2), which was raisedagainst native FMDV 3D^(pol) and specifically binds an epitope of FMDV3D^(pol), and MAb F8B (Accession No. 130514-01, deposited May 13, 2014,International Depositary Authority of Canada, 1015 Arlington Street,Winnipeg, Manitoba, Canada R3C 3R2), which was raised against FMDV 3Bprotein and specifically binds an epitope of FMDV 3B, each showed highreactivity against A24WT protein. However, MAb F32-44 and MAb F8B didnot react with the BRV2 3D^(pol) or the mutant A₂₄LL3D_(YR), andA₂₄LL3B_(PVKV)3D_(YR) viral counterparts, i.e., these antibodies did notspecifically bind to epitopes of BRV2 3D^(pol) or BRV2 3B or mutantcounterparts of 3D^(pol) or 3B, suggesting that these antigenic markersmight be of significant value as a DIVA diagnostic tool.

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Mutations introduced in the coding regions for 3B and 3D^(pol) using thepA₂₄Cru full-length plasmid produced viruses (A₂₄WT3B_(PVKV)3D_(YR))that did not react with MAbs F32-44 and F8B but showed similar plaquephenotypes and tissue culture propagation properties as the parentalA₂₄WT virus. Moreover studies in cattle and swine demonstrated thatA₂₄WT3D_(YR) and A₂₄WT3B_(PVKV)3D_(YR) mutants are highly pathogenic andable to spread the disease to contact animals as the A₂₄WT, furthersuggesting that the introduced mutation themselves, did notsignificantly affect virulence. The mutations introduced into the 3B and3D^(pol) appeared to be stable not only in tissue culture (unchangedeven after 15 serial passage sin BHK-21) but also in animals.

Reduced virulence is a critical aspect to be addressed in developing asafe vaccine to be produced in an area free of the disease. Attenuationof the double negative marker virus was achieved by manipulating thegenome to eliminate the L^(pro) coding sequence, which is known to beinvolved in FMDV pathogenesis in vivo (see the Introduction), Here(A₂₄LL, A₂₄LL3D_(YR), A₂₄LL3B_(PVKV)3D_(YR)) and in previous studies(A₁₂LLV2), it has been shown that deletion of the FMDV leader proteinasecoding sequence created viruses that maintained the ability to infectBHK-21 cells but display low virulence for cattle or pigs (Almeida etal. 1998. Virus Res. 55:49-60; Brown et al., supra; Chinsangaram et al.,supra; Mason et al. 1997, supra). Animals infected with A₂₄LL3D_(YR) bythe aerosol route (cattle) or by direct inoculation of A₂₄LL3D_(YR),A₂₄LL3B_(PVKV)3D_(YR), A-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) or Asia1-A₂₄LL3B_(PVKV)3D_(YR) viruses in the feet (swine) demonstrated thatthe prototype virus candidates are highly attenuated for clinicaldisease and unable to spread virus to contact animals on bothsusceptible livestock models. Thus, given the reduced replication ofA₂₄LL3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) as well as with chimericA-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) or Asia 1-A₂₄LL3B_(PVKV)3D_(YR)viruses in the natural host and the lack of transmission to in-contactanimals, it would appear highly unlikely that virus transmission thatleads to clinical disease could occur under field conditions as aresults of incomplete inactivation of this marker vaccine or due tovirus leaking from the manufacturing laboratory.

Animals infected with the single and double marker FMDV produced in thebackbone of a leader-containing genome (A₂₄WT3D_(YR) andA₂₄WT3B_(PVKV)3D_(YR)) developed a serological antibody response that,when analyzed at 21 dpi, allow a differentiation relative to theserologic profile observed for wild-type infected animals. This isparticularly important since the FMDV 3D^(pol) protein is known tostimulate a strong humoral and cellular immune response in the host atearly times (Collen et al. 1998. Virus Res. 56:125-133; Cowan andGraves, supra). In the cELISA utilized in this study, the 3D^(pol)antigen is captured to the solid phase, then, the ability of test serato inhibit the binding of the MAb F32-44 to the antigen is evaluated.The procedure, originally developed by Yang et al. (2007a, supra) isused for the detection of antibodies against different serotypes ofFMDV. Likewise, an in house cELISA based on competition with MAb F8B(raised against 3B protein) was used to detect serological responses tothe epitope contained in the 3B viral protein. Because mutantA₂₄LL3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) viruses lack epitopes that arepresent in the parental virus (on the basis of which cELISA wereestablished; for 3D^(pol), see FIG. 4 and for 3B, see FIG. 7), our workprovides a potential DIVA companion test to be used in conjunction withthese marker FMDV vaccine candidates. Our data support the importance ofthe antigenic site contained in the FMDV 3D^(pol)/16-32 peptide anddemonstrate that this immunodominant epitope was effectively removed inthe mutant viruses.

Killed-FMDV vaccines are presently commercially available and have beenshown to be safe and effective for the control of FMD. The killed-virusvaccine is prepared from virus grown in BHK-21 cells, ischemically-inactivated (BEI), and adjuvant is added to the viralproduct. We have demonstrated that the BEI-inactivated markerA₂₄LL3D_(YR) vaccine elicited an immune response that completelyprotected cattle from clinical disease after direct inoculation. Theseresults are similar to those observed when animals were immunized with acommercial polyvalent FMD vaccine with a standard antigen payload. Thehigh level of protection against live virus challenge was achieved inanimals that received one dose of either the experimental or commercialvaccines. The analysis of the humoral response against FMDV revealedthat both vaccine formulations were able to induce detectable levels ofneutralizing antibodies before challenge. Although we did not detect asignificant antibody response against NSPs after single dose ofvaccination (data not shown), animals infected with the A₂₄WT3D_(YR)virus developed good antibody responses to NSPs lacking recognition tothe marker epitope (see FIG. 4). In the field practice, multiple dosesof inactivated FMDV vaccines are applied for the control and preventionof FMD, and under this regimen, antibodies against 3D^(pol) are commonlydetected (Silberstein et al. 1997. Arch. Virol. 142:795-805). Since thetarget epitopes are absent from the A₂₄LL3B_(PVKV)3D_(YR) platform,antibodies against these epitopes will not develop in vaccinated animalsdespite multiple immunizations (every 6 months), a common fieldpractice. Therefore, this strategy will be feasible in the fieldpractice. In addition to the above demonstrated characteristics that aredesirable for any vaccine candidate, the A₂₄LL3D_(YR) andA₂₄LL3B_(PVKV)3D_(YR) marker viruses were also shown to be attenuated inthe natural host, but they grow well in BHK-21 cells. This virusproperty offers the advantage to reduce the risk of outbreaks originatedby escape of highly virulent FMDVs from vaccine manufacturingfacilities, where high load of viruses are handled. In addition to thedistinctive serological profile elicited by the marker viruses, themutant viruses can be differentiated from field FMDVs by geneticmethods.

Considering the high economic damage that FMD can elicit on livestock(see review by Sutmoller and Olascoaga (Sutmoller et al. 2003, supra),current vaccination programs are now supporting the “vaccinate-to-live”policy for FMD outbreaks. In this scenario, the rational design ofnegative marker (A₂₄LL3D_(YR), and A₂₄LL3B_(PVKV)3D_(YR) or chimericviruses such as A-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) orAsia1-A₂₄LL3B_(PVKV)3D_(YR)) vaccine candidates, capable to elicit anantibody response that can be differentiated from the response inducedby the wild-type virus, and used in conjunction with a companion DIVAtest (cELISA), could assist in FMD control measurements and support thedifferentiation of infected versus vaccinated animals.

Production and manipulation of the isolated polynucleotide moleculesdescribed herein are within the skill in the art and can be carried outaccording to recombinant techniques described, among other places, inSambrook et al. 1989. Molecular Cloning: A Laboratory Manual, 2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Inniset al. (eds). 1995. PCR Strategies, Academic Press, Inc., San Diego,which are incorporated herein by reference.

The subject invention provides vectors comprising genetically modifiednucleic acid sequences that encode genetically modified infectious RNAmolecules that encode genetically modified Foot and Mouth DiseaseViruses.

In particular, the subject invention provides isolated polynucleotidemolecules encoding genetically modified infectious RNA molecules thatencode genetically modified FMD viruses; namely, the vaccine candidateviruses A₂₄LL3D_(YR), and A₂₄LL3B_(PVKV)3D_(YR) which harbor negativeantigenic markers for potential DIVA capabilities, encoded in either the3D^(pol) alone or 3B and 3D^(pol) combined, respectively.

It is understood that terms herein referring to nucleic acid moleculessuch as “isolated polynucleotide molecule” and “nucleotide sequenceinclude both DNA and RNA molecules and include both single-stranded anddouble-stranded molecules whether it is natural or synthetic origin.

For example, SEQ ID NO:1 is a DNA sequence corresponding to thegenetically modified RNA genome of a genetically modified FMDV. Thus, aDNA sequence complementary to the DNA sequence set forth in SEQ ID NO:1is a template for, i.e. is complementary to or “encodes”, the RNA genomeof the FMDV virus (i.e., RNA that encodes the FMDV).

Furthermore, when reference is made herein to sequences homologous to asequence in the Sequence Listing, it is to be understood that sequencesare homologous to a sequence corresponding to the sequence in theSequence Listing and to a sequence complementary to the sequence in theSequence Listing.

An “infectious RNA molecule”, for purposes of the present invention, isan RNA molecule that encodes the necessary elements for viralreplication, transcription, and translation into a functional virion ina suitable host cell, provided, if necessary, with a peptide or peptidesthat compensate for any genetic modifications, e.g. sequence deletions,in the RNA molecule.

An “isolated infectious RNA molecule” refers to a composition of mattercomprising the aforementioned infectious RNA molecule purified to anydetectable degree from its naturally occurring state, if such RNAmolecule does indeed occur in nature. Likewise, an “isolatedpolynucleotide molecule” refers to a composition of matter comprising apolynucleotide molecule of the present invention purified to anydetectable degree from its naturally occurring state, if any.

For purposes of the present invention, two nucleotide (RNA or DNA)sequences are substantially homologous when at least 80% (preferably atleast 85% and most preferably 90%) of the nucleotides match over thedefined length of the sequence using algorithms such as CLUSTRAL orPHILIP. Sequences that are substantially homologous can be identified ina Southern hybridization experiment under stringent conditions as isknown in the art. See, for example, Sambrook et al., supra. Sambrook etal. describe highly stringent conditions as a hybridization temperature5-10° C. below the T_(m) of a perfectly matched target and probe; thus,sequences that are “substantially homologous” would hybridize under suchconditions.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of nucleotides thatdo not substantially affect the functional properties of the resultingtranscript. It is therefore understood that the invention encompassesmore than the specific exemplary nucleotide or amino acid sequences andincludes functional equivalents thereof. Alterations in a nucleic acidfragment that result in the production of a chemically equivalent aminoacid at a given site, but do not affect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. A method of selecting an isolated polynucleotide that affectsthe level of expression of a polypeptide in a virus or in a host cell(eukaryotic, such as plant, yeast, fungi, or algae; prokaryotic, such asbacteria) may comprise the steps of: constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; introducing the isolated polynucleotide or theisolated chimeric gene into a host cell; measuring the level of apolypeptide in the host cell containing the isolated polynucleotide; andcomparing the level of a polypeptide in the host cell containing theisolated polynucleotide with the level of a polypeptide in a host cellthat does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by DNA-DNA, DNA-RNA, or RNA-RNA hybridization underconditions of stringency as is well understood by those skilled in theart (1985. Nucleic Acid Hybridization, Hames and Higgins, Eds., IRLPress, Oxford, U.K.). Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.

Thus, isolated sequences that encode a modified FMDV, i.e., A₂₄LL3D_(YR)(SEQ ID NO:1) and/or A₂₄LL3B_(PVKV)3D_(YR) (SEQ ID NO:3), and whichhybridize under stringent conditions, as described herein, to themodified FMDVs, the A₂₄LL3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) sequencesdisclosed herein, i.e., SEQ ID NO:1, SEQ ID NO:3, or to fragmentsthereof, are encompassed by the present invention. Fragments of anucleotide sequences that are useful as hybridization probes may notencode fragment proteins retaining biological activity.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988. CABIOS 4:11-17), the local homology algorithmof Smith et al. (1981. Adv. Appl. Math. 2:482); the homology alignmentalgorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48:443-453); thesearch-for-similarity-method of Pearson and Lipman (1988. Proc. Natl.Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990.Proc. Natl. Acad. Sci. USA 87:2264), modified as in Karlin and Altschul(1993. Proc. Natl. Acad. Sci. USA 90:5873-5877).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90%, mostpreferably at least 95% sequence identity compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes normally means sequence identity of at least 80%, preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST. In general, a sequence of ten ormore contiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification and isolation. Inaddition, short oligonucleotides of 12 or more nucleotides may be use asamplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises a nucleotide sequence thatwill afford specific identification and/or isolation of a nucleic acidfragment comprising the sequence. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for purposes known tothose skilled in this art. Accordingly, the instant invention comprisesthe complete sequences as reported in the accompanying Sequence Listing,as well as substantial portions at those sequences as defined above.

By “variants” substantially similar sequences are intended. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode theA₂₄LL3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) sequences, the amino acidsequences of the invention. Naturally occurring allelic variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR), atechnique used for the amplification of specific DNA segments.Generally, variants of a particular nucleotide sequence of the inventionwill have generally at least about 90%, preferably at least about 95%and more preferably at least about 98% sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms described elsewhere herein.

By “variant protein” a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the present invention are biologically active, that isthey possess the desired biological activity, that is, a modifiedA₂₄LL3D_(YR) and/or A₂₄LL3B_(PVKV)3D_(YR) activity. Such variants mayresult from, for example, genetic polymorphism or from humanmanipulation. Biologically active variants of modified A₂₄LL3D_(YR) andA₂₄LL3B_(PVKV)3D_(YR) activities of the invention will have at leastabout 90%, preferably at least about 95%, and more preferably at leastabout 98% sequence identity to the amino acid sequence for the nativeprotein as determined by sequence alignment programs described elsewhereherein. A biologically active variant of a protein of the invention maydiffer from that protein by as few as 1-15 amino acid residues, or even1 amino acid residue.

The polypeptides of the invention may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Novel proteins having properties of interest may be createdby combining elements and fragments of proteins of the presentinvention, as well as with other proteins. Methods for suchmanipulations are generally known in the art. Thus, the genes andnucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant forms. Likewise, the proteins ofthe invention encompass naturally occurring proteins as well asvariations and modified forms thereof. Such variants will continue topossess the desired modified FMDV A₂₄LL3D_(YR) and/orA₂₄LL3B_(PVKV)3D_(YR) activities. Obviously, the mutations that will bemade in the DNA encoding the variant must not place the sequence out ofreading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays where the effects of modifiedFMDV A₂₄LL3D_(YR) and/or A₂₄LL3B_(PVKV)3D_(YR) can be observed.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein.

It is furthermore to be understood that the isolated polynucleotidemolecules and the isolated RNA molecules of the present inventioninclude both synthetic molecules and molecules obtained throughrecombinant techniques, such as by in vitro cloning and transcription.

As used herein, the term “FMD” encompasses disease symptoms in cattleand swine caused by a FMDV infection. Examples of such symptoms include,but are not limited to, vesicles in the mouth, and on the feet. As usedherein, a FMDV that is “unable to produce FMD” refers to a virus thatcan infect a pig, but which does not produce any disease symptomsnormally associated with a FMD infection in the pig, or produces suchsymptoms, but to a lesser degree, or produces a fewer number of suchsymptoms, or both.

The terms “porcine” and “swine” are used interchangeably herein andrefer to any animal that is a member of the family Suidae such as, forexample, a pig. “Mammals” include any warm-blooded vertebrates of theMammalia class, including humans.

The terms “foot and mouth disease virus” and “FMDV”, as used herein,unless otherwise indicated, mean any strain of FMD viruses.

The term “open reading frame”, or “ORF”, as used herein, means theminimal nucleotide sequence required to encode a particular FMDV proteinwithout an intervening stop codon.

Terms such as “suitable host cell” and “appropriate host cell”, unlessotherwise indicated, refer to cells into which RNA molecules (orisolated polynucleotide molecules or viral vectors comprising DNAsequences encoding such RNA molecules) of the present invention can betransformed or transfected. “Suitable host cells” for transfection withsuch RNA molecules, isolated polynucleotide molecules, or viral vectors,include mammalian, particularly bovine and porcine cells, and aredescribed in further detail below.

A “functional virion” is a virus particle that is able to enter a cellcapable of hosting a FMDV, and express genes of its particular RNAgenome (either an unmodified genome or a genetically modified genome asdescribed herein) within the cell. Cells capable of hosting a FMDVinclude baby hamster kidney, strain 21, cells (BHK-21) and BHK-α_(V)β₆cells expressing bovine α_(V)β₆ integrin. Other mammalian cells,especially other bovine and porcine cells, may also serve as suitablehost cells for FMDV virions.

The isolated polynucleotide molecules of the present invention encodeFMD viruses that can be used to prepare live attenuated vaccines usingart-recognized methods for protecting cattle and swine from infection bya FMDV, as described in further detail below. Furthermore, theseisolated polynucleotide molecules are useful because they can be mutatedusing molecular biology techniques to encode genetically-modified FMDviruses useful, inter alia, as vaccines for protecting cattle and swinefrom FMD infection. Such genetically-modified FMD viruses, as well asvaccines comprising them, are described in further detail below.

Accordingly, the subject invention further provides a method for makinga genetically modified FMDV, which method comprises mutating the DNAsequence encoding an infectious RNA molecule which encodes the FMDV asdescribed above, and expressing the genetically modified FMDV using asuitable expression system. A FMDV, either wild-type or geneticallymodified, can be expressed from an isolated polynucleotide moleculeusing suitable expression systems generally known in the art, examplesof which are described in this application. For example, the isolatedpolynucleotide molecule can be in the form of a plasmid capable ofexpressing the encoded virus in a suitable host cell in vitro.

The term “genetically modified”, as used herein and unless otherwiseindicated, means genetically mutated, i.e. having one or morenucleotides replaced, deleted and/or added. Polynucleotide molecules canbe genetically mutated using recombinant techniques known to those ofordinary skill in the art, including by site-directed mutagenesis, or byrandom mutagenesis such as by exposure to chemical mutagens or toradiation, as known in the art.

The subject invention further provides an isolated polynucleotidemolecule comprising a DNA sequence encoding an infectious RNA moleculewhich encodes a genetically modified FMDV that is unable to produce FMDin cattle and/or swine, wherein the DNA sequence encoding the infectiousRNA molecule encoding said modified FMDV A₂₄LL3D_(YR) comprises SEQ IDNO:1, said modified FMDV A₂₄LL3B_(PVKV)3D_(YR) comprises SEQ ID NO:3 orsequences homologous thereto, contain one or more mutations thatgenetically disable the encoded FMDV in its ability to produce FMD.“Genetically disabled” means that the FMDV is unable to produce FMD in abovine or swine animal infected therewith.

In one embodiment, the genetically modified FMDV disabled in its abilityto cause FMD is able to elicit an effective immunoprotective responseagainst infection by FMDV in cattle or swine. Accordingly, the subjectinvention also provides an isolated polynucleotide molecule comprising aDNA sequence encoding an infectious RNA molecule which encodes a FMDVthat is genetically modified such that when it infects cattle and/orswine it: a) is unable to produce FMD in the animal, and b) is able toelicit an effective immunoprotective response against infection by aFMDV in the animal, wherein the DNA sequence encoding said modified FMDVA₂₄LL3D_(YR) comprises SEQ ID NO:1, said modified FMDVA₂₄LL3B_(PVKV)3D_(YR) comprises SEQ ID NO:3, or sequences homologousthereto, contain one or more mutations that genetically disable theencoded FMDV in its ability to produce FMD.

The term “immune response” for purposes of this invention means theproduction of antibodies and/or cells (such as T lymphocytes) that aredirected against, or assist in the decomposition or inhibition of, aparticular antigenic epitope or particular antigenic epitopes. Thephrases “an effective immunoprotective response”, “immunoprotection”,and like terms, for purposes of the present invention, mean an immuneresponse that is directed against one or more antigenic epitopes of apathogen so as to protect against infection by the pathogen in avaccinated animal. For purposes of the present invention, protectionagainst infection by a pathogen includes not only the absoluteprevention of infection, but also any detectable reduction in the degreeor rate of infection by a pathogen, or any detectable reduction in theseverity of the disease or any symptom or condition resulting frominfection by the pathogen in the vaccinated animal as compared to anunvaccinated infected animal. An effective immunoprotective response canbe induced in animals that have not previously been infected with thepathogen and/or are not infected with the pathogen at the time ofvaccination. An effective immunoprotective response can also be inducedin an animal already infected with the pathogen at the time ofvaccination.

An “antigenic epitope” is, unless otherwise indicated, a molecule thatis able to elicit an immune response in a particular animal or species.Antigenic epitopes are proteinaceous molecules, i.e. polypeptidesequences, optionally comprising non-protein groups such as carbohydratemoieties and/or lipid moieties.

The genetically modified FMD viruses encoded by the above-describedisolated polynucleotide molecules are, in one embodiment, able to elicitan effective immunoprotective response against infection by a FMDV. Suchgenetically modified FMD viruses are preferably able to elicit aneffective immunoprotective response against any strain of FMD viruses.

In one embodiment, the mutation or mutations in the isolatedpolynucleotide molecule encoding the genetically disabled FMDV arenon-silent and occur in one or more open reading frames of thenucleotide sequence encoding the FMDV.

As used herein, unless otherwise indicated, “coding regions” refer tothose sequences of RNA from which FMDV proteins are expressed, and alsorefer to cDNA that encodes such RNA sequences. Likewise, “ORFs” referboth to RNA sequences that encode FMDV proteins and to cDNA sequencesencoding such RNA sequences.

Determining suitable locations for a mutation or mutations that willencode a FMDV that is genetically disabled so that it is unable toproduce FMD yet remains able to elicit an effective immunoprotectiveresponse against infection by a FMDV and which can differentiate anaturally infected animal from a vaccinated animal can be made based onSEQ ID NO:1 and/or SEQ ID NO:3 provided herein. One of ordinary skillcan refer to the sequence of the infectious cDNA clone of FMDV providedby this invention, make sequence changes which will result in a mutationaltering the leader sequence as well as sequences within 3D^(pol) and3B, and test the viruses encoded thereby for their abilities to produceFMD in swine, to elicit an effective immunoprotective response againstinfection by a FMDV, and to make possible the differentiation ofinfected vs. vaccinated animals. In so doing, one of ordinary skill canrefer to techniques known in the art and also those described and/orexemplified herein.

For example, an ORF of the sequence encoding the infectious RNA moleculeencoding the FMDV can be mutated and the resulting genetically modifiedFMDV tested for its ability to cause FMD.

In a further preferred embodiment, an antigenic epitope of thegenetically modified FMDV of the present invention results in a negativemarker. Such isolated polynucleotide molecules and the FMD viruses theyencode are useful, inter alia, for studying FMD infections in cattle andswine, determining successfully vaccinated cattle and swine, and/or fordistinguishing vaccinated cattle and swine from cattle and swineinfected by a wild-type FMDV. Preferably, such isolated polynucleotidemolecules further contain one or more mutations that genetically disablethe encoded FMDV in its ability to produce FMD, and more preferably areable to elicit an effective immunoprotective response in bovine andporcine animals against infection by a FMDV.

Antigenic epitopes that are detectable, and the sequences that encodethem, are known in the art. Techniques for detecting such antigenicepitopes are also known in the art and include serological detection ofantibody specific to the heterologous antigenic epitope by means of, forexample, Western blot, ELISA, or fluorescently labeled antibodiescapable of binding to the antibodies specific to the heterologousantigenic epitope. Techniques for serological detection useful inpracticing the present invention can be found in texts recognized in theart, such as Coligan, J. E., et al. (eds), 1998, Current Protocols inImmunology, John Willey & Sons, Inc., which is hereby incorporated byreference in its entirety. Alternatively, the antigenic epitope itselfcan be detected by, for example, contacting samples that potentiallycomprise the antigenic epitope with fluorescently-labeled antibodies orradioactively-labeled antibodies that specifically bind to the antigenicepitopes.

The present invention further provides an isolated polynucleotidemolecule comprising a DNA sequence encoding an infectious RNA moleculewhich encodes a genetically modified FMDV that detectably lacks FMDVantigenic epitope, wherein the DNA sequence encoding the RNA moleculeencoding the modified FMDV is SEQ ID NO:1, SEQ ID NO:3, or sequenceshomologous thereto, except that it lacks one or more nucleotidesequences encoding a detectable FMDV antigenic epitope. Such isolatedpolynucleotide molecules are useful for distinguishing between cattleand/or swine infected with a recombinant FMDV of the present inventionand cattle and/or swine infected with a wild-type FMDV. For example,animals vaccinated with killed, live or attenuated FMDV encoded by suchan isolated polynucleotide molecule can be distinguished from animalsinfected with wild-type FMDV based on the absence of antibodies specificto the missing antigenic epitope, or based on the absence of theantigenic epitope itself: If antibodies specific to the missingantigenic epitope, or if the antigenic epitope itself, are detected inthe animal, then the animal was exposed to and infected by a wild-typeFMDV. Means for detecting antigenic epitopes and antibodies specificthereto are known in the art, as discussed above. Preferably, such anisolated polynucleotide molecule further contains one or more mutationsthat genetically disable the encoded FMDV in its ability to produce FMD.More preferably, the encoded virus remains able to elicit an effectiveimmunoprotective response against infection by a FMDV.

Vaccines of the present invention can be formulated following acceptedconvention to include acceptable carriers for animals, including humans(if applicable), such as standard buffers, stabilizers, diluents,preservatives, and/or solubilizers, and can also be formulated tofacilitate sustained release. Diluents include water, saline, dextrose,ethanol, glycerol, and the like. Additives for isotonicity includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin, among others. Other suitablevaccine vehicles and additives, including those that are particularlyuseful in formulating modified live vaccines, are known or will beapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention can further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Non-limiting examples of adjuvants that can beused in the vaccine of the present invention include the RIBI adjuvantsystem (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminumhydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as,e.g., Freund's complete and incomplete adjuvants, Block copolymer(CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.),SAF-M (Chiron, Emeryville Calif.), AMPHIGEN® adjuvant, saponin, Quil Aor other saponin fraction, monophosphoryl lipid A, and Avridinelipid-amine adjuvant. Non-limiting examples of oil-in-water emulsionsuseful in the vaccine of the invention include modified SEAM62 and SEAM1/2 formulations. Modified SEAM62 is an oil-in-water emulsion containing5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICISurfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5%(v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v)lecithin. Modified SEAM 1/2 is an oil-in-water emulsion comprising 5%(v/v) squalene, 1% (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween® 80detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/mlcholesterol. Other immunomodulatory agents that can be included in thevaccine include, e.g., one or more interleukins, interferons, or otherknown cytokines.

Vaccines of the present invention can optionally be formulated forsustained release of the virus, infectious RNA molecule, plasmid, orviral vector of the present invention. Examples of such sustainedrelease formulations include virus, infectious RNA molecule, plasmid, orviral vector in combination with composites of biocompatible polymers,such as, e.g., polylactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, collagen and the like. The structure,selection and use of degradable polymers in drug delivery vehicles havebeen reviewed in several publications, including Domb et al. 1992.Polymers for Advanced Technologies 3: 279-292, which is incorporatedherein by reference. Additional guidance in selecting and using polymersin pharmaceutical formulations can be found in texts known in the art,for example M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymersas Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences,Vol. 45, M. Dekker, NY, which is also incorporated herein by reference.Alternatively, or additionally, the virus, plasmid, or viral vector canbe microencapsulated to improve administration and efficacy. Methods formicroencapsulating antigens are well-known in the art, and includetechniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No.3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat.No. 4,744,933; U.S. Pat. No. 5,132,117; and International PatentPublication WO 95/28227, all of which are incorporated herein byreference.

Liposomes can also be used to provide for the sustained release ofvirus, plasmid, or viral vector. Details concerning how to make and useliposomal formulations can be found in, among other places, U.S. Pat.No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S.Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050;and U.S. Pat. No. 5,009,956, all of which are incorporated herein byreference.

An effective amount of any of the above-described vaccines can bedetermined by conventional means, starting with a low dose of virus,plasmid or viral vector, and then increasing the dosage while monitoringthe effects. An effective amount may be obtained after a singleadministration of a vaccine or after multiple administrations of avaccine. Known factors can be taken into consideration when determiningan optimal dose per animal. These include the species, size, age andgeneral condition of the animal, the presence of other drugs in theanimal, and the like. The actual dosage is preferably chosen afterconsideration of the results from other animal studies.

One method of detecting whether an adequate immune response has beenachieved is to determine seroconversion and antibody titer in the animalafter vaccination. The timing of vaccination and the number of boosters,if any, will preferably be determined by a doctor or veterinarian basedon analysis of all relevant factors, some of which are described above.

The effective dose amount of virus, infectious RNA molecule, plasmid, orviral vector, of the present invention can be determined using knowntechniques, taking into account factors that can be determined by one ofordinary skill in the art such as the weight of the animal to bevaccinated. The dose amount of virus of the present invention in avaccine of the present invention preferably ranges from about 10¹ toabout 10⁹ pfu (plaque forming units), more preferably from about 10² toabout 10⁸ pfu, and most preferably from about 10³ to about 10⁷ pfu. Thedose amount of a plasmid of the present invention in a vaccine of thepresent invention preferably ranges from about 0.1 g to about 100 mg,more preferably from about 1 μg to about 10 mg, even more preferablyfrom about 10 μg to about 1 mg. The dose amount of an infectious RNAmolecule of the present invention in a vaccine of the present inventionpreferably ranges from about 0.1 μg to about 100 mg, more preferablyfrom about 1 μg to about 10 mg, even more preferably from about 10 μg toabout 1 mg. The dose amount of a viral vector of the present inventionin a vaccine of the present invention preferably ranges from about 10¹pfu to about 10⁹ pfu, more preferably from about 10² pfu to about 10⁸pfu, and even more preferably from about 10³ to about 10⁷ pfu. Asuitable dosage size ranges from about 0.5 ml to about 10 ml, and morepreferably from about 1 ml to about 5 ml.

In summary, our studies provide a recombinant viral-vectored vaccineplatform comprising a genetically modified FMDV comprising deletion ofthe LP^(pro) coding sequence, mutations (negative markers) introduced intwo non-structural viral proteins resulting in the elimination of twoantigenic epitopes recognized by specific antibodies, one located in 3Band the other in 3D, thus providing targets for DIVA (Differentiation ofnaturally Infected from Vaccinated Animals) serological tests, andinclusion of unique restriction endonuclease sites to facilitate thereplacement of the capsid region; and the genetically modified FMDV hasbeen chemically-inactivated. The rationally designed attenuated FMDVvaccine production platform can be used to manufacture inactivatedantigen vaccine that is effective to protect an animal from clinical FMDwhen challenged with virulent FMDV and is a marker vaccine which allowsa serological distinction between vaccinated animals and animalsinfected with FMDV.

EXAMPLES

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein only to further illustrate the invention and are not intended tolimit the scope of the invention as defined by the claims.

Example 1 Viruses and Cell Cultures

FMDV type A₂₄ Cruzeiro was derived from the infectious cDNA clonepA₂₄Cru (called here for simplicity A₂₄WT, (Rieder et al. 2005. J.Virol. 79:12989-12998). A plasmid containing the bovine rhinovirus type2 (pBRV2, accession number EU236594) sequence from poly[C] to poly[A]described previously (Hollister et al., supra) was used as a source ofbovine rhinovirus genetic material. Baby hamster kidney strain 21, clone13, cell line (BHK-21) was maintained in Eagle's basal medium (BME)(Life Technologies, Gaithersburg, Md.) supplemented with 10% bovine calfserum (BCS) (Hyclone, South Logan, Utah), 10% Tryptose phosphate broth,and antibiotic/antimycotic. Monolayers of a continuous bovine kidneycell line (LFBK, (Swaney, L. M. 1988. Vet. Microbiol. 18:1-14) weregrown in Eagle's minimal essential medium (MEM) containing 10% fetalcalf serum (Hyclone, South Logan, Utah) and antibiotic/antimycotic.Swine kidney cells (de Castro, M. P. 1964. Arq. Inst. Biol. (Sao Paulo)31:63-78.) were propagated in Dulbecco's modified Eagle's medium (D-MEM)supplemented with 10% FBS and antibiotic/antimycotic. BHK-α_(V)β₆ is astable cell line expressing the bovine α_(V)β₆ integrin, propagated inBME containing 10% bovine calf serum (Hyclone, South Logan, Utah), withthe addition of G418 and Zeocin (Invitrogen), and has been previouslydescribed (Duque et al. 2004. J. Virol. 78:9773-9781). Cells were grownat 37° C. in a humidified with 5% CO₂ atmosphere.

Example 2 Non-Structural Protein 3D^(pol): Immunohistochemistry andRadioimmunoprecipitation Assays

Our previous studies have identified several invariant amino acids inthe 3D^(pol) protein of the closely-related FMDV and BRV2 3D^(pol)viruses (Hollister et al., supra). To further determine if thesesequence similarities result in the display of similar (shared) epitopesbetween the corresponding 3D^(pol) of these viruses, we carried out aradioimmuoprecipitation (RIP) assay using specific polyclonal ormonoclonal antibodies (MAb) specific for FMDV 3D^(pol). To this end,FMDV or BRV2 transcript RNAs derived from pA₂₄Cru and pBRV2 weretranslated in vitro in the presence of ³⁵S-methonine and then theextracts were subjected to RIP using FMDV-specific anti-3D^(pol) rabbitpolyclonal sera or the MAbs F19-59 and F32-44 directed against the FMDVnon-structural protein 3D^(pol) and partially characterized by Yang etal. (2007a, supra).

Cell monolayers grown in 6-well plates were infected with virus at anMOI=1 and 6 h later the infected cells were fixed with coldacetone:methanol (50/50) mix for 20 min followed by two washes with PBS.Fixed cells were immunoperoxidase-stained using FMDV-specific MAbfollowing the manufacturer's instructions of the Vectastain ABC AlkalinePhosphatase Kit from Vector labs. Reactivity of antibodies to 3D^(pol)was also measured using radioimmunoprecipitation (RIP) assays performedas described by Rieder et al. (1994. J. Virol. 68:7092-7098).

As shown in FIG. 1, SDS-PAGE analysis of the radioimmunoprecipitatesrevealed a strong reaction for MAb F19-59 and the anti 3D^(pol) rabbitpolyclonal sera with both sources (FMDV and BRV2) of viral 3D^(pol)proteins. In contrast, MAb F32-44 reacted only with FMDV 3D^(pol) and inrepeated RIPs attempts it failed to recognize the BRV-2 protein (FIG.1). These results indicate that BRV2 3D^(pol) lacks the epitope found onthe FMDV 3D^(pol) protein that is recognized by MAb F32-44.

Example 3 Derivation of A₂₄LL Negative Marker FMDV Viruses

Two mutant plasmids designated pA₂₄WT3D_(YR) and pA₂₄LL3D_(YR) werederived by site-directed mutagenesis using either full-length infectiousclone of FMDV pA₂₄Cru (Rieder et al. 2005, supra) or the backbone of theleader-deleted pA₂₄LL infectious cDNA clones. Plasmids p A₂₄WT3D_(YR)and pA₂₄LL3D_(YR) were engineered with a substitution in 3D^(pol) foundin BRV2 at the respective locations that would eliminates an importantantigenic epitope in 3D^(pol); His₂₇ was replaced by Tyr (H₂₇>Y) andAsn₃₁ was changed to the basic amino acid Arg (N₃₁>R) (FIG. 2). Thus,both A₂₄WT3D_(YR) and A₂₄LL3D_(YR) have the His₂₇ and Asn₃₁ of the WTFMDV replaced by the BRV2 amino acids Tyr and Arg, respectively. The3D^(pol) antigen of WT FMDV has the amino acid sequence of SEQ ID NO:5;the mutant 3D^(pol) comprising the substitution of the BRV2 amino acidshas the amino acid sequence of SEQ ID NO:6 and is found in A₂₄WT3D_(YR)and A₂₄LL3D_(YR) viruses. Infectious RNA were in vitro transcribed frominfectious cDNA clones and virus rescued from BHK-21 transfected cellsconfirmed that only the mutation at the 3D^(pol) locus encoding the YRepitope was present in the mutant viruses.

The 3D^(pol) region of pA₂₄Cru (A₂₄WT) was modified by PCR utilizingmutagenic oligonucleotides P1266(5′-ACCGTTGCGTACGGTGTGTTCCGTCCTGAGTTCGGG; SEQ ID NO:7) and P1267 (5′CCCGAACTCAGGACGGAACACACCGTACGCAAC GGT; SEQ ID NO:8) engineered tointroduce mutations at codons 27 and 31 of protein 3D^(pol) (see FIG.2). Deletion of Leader gene and introduction of FseI at the beginning ofthe coding region for the capsid viral protein VP4 and NheI site in 2Awere generated by overlap PCR fusion, created by mixing PCR-amplifiedfragments, re-amplifying through the product of the fusion of these twofragments. This was accomplished by using oligonucleotide P819(5′-CGAGCCACAGGAAGGATGGGGGCCGGCCAATCCAG; SEQ ID NO:9) P820(5′-CTGGATTGGCCGGCCCCCATCCTTCCTGTGGCTCG; SEQ ID NO:10) containing anFseI site added by silent mutation in VP4 and P/2A-NheI(s)(GACCTGCTTAAGCTAGCCGGAGACGTTGA; SEQ ID NO:11) and P/2A-NheI(a)(5′TCAACGTCTCCGGCTAGCTTAAGCAGGTC; SEQ ID NO:12) containing a silentmutation that introduces NheI in the coding region for 2A. The generatedplasmids pA₂₄Cru, pA₂₄WT3D_(YR), pA₂₄LL3D_(YR) and pA₂₄LL all contain aT7 promoter sequence in front of a hammerhead ribozyme at the 5′terminusof the S fragment of the FMDV genome, and terminates with a poly(A)tract of 15 residues and they possess a unique restriction site (SwaI)used for linearization.

A double negative epitope A₂₄WT3B_(PVKV)3D_(YR) andA₂₄LL3B_(PVKV)3D_(YR) mutant FMDVs were derived from plasmidspA₂₄WT3D_(YR), pA₂₄LL3D_(YR) respectively, lacking one of their 3B (3B₁or also known as VPg₁) proteins, and containing a substitution in 3B₂that abolished reactivity with MAb F8B (a gift from Alfonso Clavijo,National Centre for Foreign Animal Disease, Winnipeg, Manitoba, Canada).To derive these mutant plasmids, a PCR product spanning sequencesbetween the unique restriction sites SalI and AgeI were produced lacking3B₁ and harboring a substitution of 3B2 sequence RQKP at amino acids9-12 by PVKV found at similar position in bovine rhinitis-2. The cDNAtemplate for PCR corresponded to a 3B₁ deleted mutant virus A₂₄WT-5853that arose from transfection of a RNA that contained a transposoninsertion in 3B₁ at position 5853 (Pacheco et al., supra). This virushas shown to grow in vitro and produce signs of FMD in cattle similar toWT A₂₄WT virus. The sequence encoding 3B₂ was modified by PCR utilizingmutagenic oligonucleotides P-PVKVs (5′-GCCCGATGGAGAGACCAGTTAAAGTTAAAGTGAAAGCAAAAGCC; SEQ ID NO:13) and P-PVKVa (5′GGCTTTTGCTTTCACTTTAACTTTAACTGGTCTCTCCATCGGGC; SEQ ID NO:14) engineeredto introduce mutations at codons 9-12 of protein 3B₂ (also known asVPg₂, see FIG. 6), Thus, both WT3B_(PVKV) and LL3B_(PVKV) have the RQKPat amino acids 9-12 of the WT FMDV replaced by the BRV2 amino acidsPVKV, respectively. The 3B antigen of WT FMDV has the amino acidsequence of SEQ ID NO:15; the mutant 3B comprising the substitution ofthe BRV2 amino acids has the amino acid sequence of SEQ ID NO:16 and isfound in A₂₄WT3B_(PVKV)3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) viruses.

Mutagenic primers were used to introduce two new restriction sites intothe full-length cDNA of FMDV as silent mutations. Primer5′-GAATGGGGGCCGGCCAATCC AGT (SEQ ID NO:19) introduces a unique FseIrestriction site at the N-terminal of VP4. Primer5′-GACCTGCTTAAGCTAGCCGGAGACGTTGAG (SEQ ID NO:20) was used to introduce aunique NheI restriction site in 2A of the FMDV coding region.

Full-length genomic clones were linearized with SwaI and in vitrotranscribed using the T7 Megascripts system (Ambion, Austin, Tex.).Transcript RNAs were transfected into BHK-21 cells by electroporation aspreviously described (Rieder et al. 1993. J. Virol. 67:5139-5145). Thetransfected cells were seeded in 6-well plates and incubated for 24-48 hat 37 C and 5% CO₂. Virus was serially passed up to 10 times in BHK-21or up to 4 times in BHK-α_(V)β₆. Virus stocks at passage 4 inBHK-α_(V)β₆ cells to be used in animal experiments and for theproduction of inactivated vaccine were entirely sequenced and stored at−70 C. Virus titers were determined by plaque assays as described below.

In vitro growth kinetics of A₂₄LL, A₂₄WT3D_(YR) and A₂₄LL3D_(YR) mutantsrelative to parental A₂₄WT virus were determined using a highmultiplicity of infection (MOI of 5) in BHK-21, LFBK cells and IBRS-2cells (FIG. 3A). While the titers of the parental and A₂₄WT3D_(YR)viruses peaked at about 6-8 hours post-inoculation (hpi) and remainedthe same over 24 h period of time in BHK-21 cells, mutants A₂₄LL,A₂₄LL3D_(YR) exhibited a 10-fold decrease in the final virus yield. Thegrowth restriction for leader deleted A₂₄LL and A₂₄LL3D_(YR) viruses wasmore noticeable in cells of bovine or swine origin (˜2.5-3 logs lowertiters compared to WT viruses in these cells). As shown in FIG. 3B, theplaque morphologies of A₂₄LL and pA₂₄LL3D_(YR) viruses were slightlysmaller and more homogeneous in size (FIG. 3B) than A₂₄WT andA₂₄WT3D_(YR) viruses.

Finally, the antigenic profile of mutant and parental viruses wereexamined by IP assays using MAbs F19-59 and F32-44, as described above(FIG. 3C). While immunoreactivity with MAbs F19-59 and F32-44 werepositive with A₂₄WT virus, the reactivity was completely abolished inA₂₄LL3D_(YR) and A₂₄WT3D_(YR) 3D^(pol) mutant viruses for F32-44. Theseresults indicate that mutation of the 3D^(pol) epitope (two amino acidsreplacement) affected the ability of FMDV to be recognized by MAbsF32-44 but not by F19-59.

Example 4 Rescue of Parental and Mutant Viruses, Viral Growth, andPlaque Assays; In Vitro Characterization of Double Negative MarkerA₂₄LL3B_(PVKV)3D_(YR) FMDV

Infectious RNAs were in vitro transcribed from full-length cDNA clonesof FMDV strain A₂₄WT or double marker mutants A₂₄WT3B_(PVKV)3D_(YR) andA₂₄LL3B_(PVKV)3D_(YR) and used to transfect BHK-21 cells, Mutantsreferred to as 3B_(PVKV)3D_(YR) contain nucleotide substitutions withinthe viral proteins 3B and 3D^(pol) by those sequences found in bovinerhinitis virus type 2 at similar positions (FIG. 6A). Thesesubstitutions were designed to produce mutant viruses that no longerreact with FMDV-specific MAbs F8B (against 3B) and F32-44 (against3D^(pol)) (FIG. 6B and FIG. 2, respectively). In vitro growthcharacteristics of mutants A₂₄WT3B_(PVKV)3D_(YR) andA₂₄LL3B_(PVKV)3D_(YR) was evaluated relative to the parental virus byplaque assays in BHK-21 cells. A₂₄WT3B_(PVKV)3D_(YR) and A₂₄WT exhibiteda mix of predominantly large plaques while A₂₄LL3B_(PVKV)3D_(YR)exhibited reduced plaque morphologies (FIG. 6C). The identity andstability of 3B and 3D^(pol) epitope mutations were confirmed bynucleotide sequence analysis of virus recovered from up to 15 virusserial passages in BHK-21 cells (data not shown).

For virus growth curves, BHK-21 monolayers were infected with A₂₄WT,A₂₄WT3D_(YR), A₂₄LL3D_(YR) or A₂₄LL at a multiplicity of infection (MOI)of 5-10 pfu/cell. After 1 h of adsorption at 37° C., monolayers wererinsed with MES buffer (Morpholine Ethane Sulfonic acid 25 mM, 145 mMNaCl, pH 5.5), then twice with PBS, followed by addition of fresh BMEcontaining no serum. At various times post-infection, viral titers weredetermined by plaque assays (Rieder et al. 1993, supra) using a 0.6% gumtragacanth overlay and incubated for 48 h at 37° C. Plates were fixed,stained with crystal violet (0.3% in Histochoice; Amresco, Solon, Ohio),and the plaques counted. Titers were expressed as plaque forming unitsper milliliter (PFU/ml) and performed in triplicates.

Example 5 Derivation of Chimeric Negative Marker FMDV Viruses

Two mutant plasmids designated, A/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) orAsia1-A₂₄LL3B_(PVKV)3D_(YR) were derived by the replacement of the A₂₄capsid with the capsid coding regions of Asia1 and Type A Turkey06 FMDVstrains, respectively. The capsids were inserted by utilizing the twounique endonuclease sites, FseI and NheI, which were engineered into thebackbone of the pA₂₄LL 3B_(PVKV)3D_(YR) infectious cDNA clone (FIG. 8A).

The P1 capsid region of FMDV Asia1/Shamir was amplified by PCR usingprimers P1629 (5′-CCACAGGAATGGGGGCCGGCCAATCCAG; SEQ ID NO:21) containinga FseI site added by silent mutation in VP4 and P1634 (5′-TCTCCGGCTAGCTTAAGCAGGTCAAAATTCAGAAGCTGCTTCTCAGGTGCAATGA; SEQ ID NO:22)containing a silent mutation that introduces NheI site in the codingregion in 2A. In addition, an internal NheI site in VP4 region wasaltered using silent mutations with primers P1690(5′-GCCTGGCAAGTTCTGCATTCAGTGG; SEQ ID NO:23) and P1691(5′-CCACTGAATGCAGAACTTGCCAGGC; SEQ ID NO:24).

The P1 capsid region of FMDV A/Turkey06 was amplified by PCR usingprimers P1629 (5′-CCACAGGAATGGGGGCCGGCCAATCCAG; SEQ ID NO:21) containinga FseI site added by silent mutation in VP4 and P1622(5′-TCTCCGGCTAGCTTAAGC AGGTCAAAATTCAGAAGTTGTTTTGCAGGTGCA; SEQ ID NO:25)containing a silent mutation that introduces NheI site in the codingregion in 2A.

The capsids of Asia and Turkey were then ligated into the backbone ofA₂₄ LL 3B_(PVKV)3D_(YR) digested with FseI and NheI to generateAsia1-A₂₄LL3B_(PVKV)3D_(YR) (SEQ ID NO: 26) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) (SEQ ID NO:27), respectively.

Full-length genomic clones were linearized with SwaI and invitro-transcribed using the T7 Megascripts system (Ambion, Austin,Tex.). Transcript RNAs were transfected into BHK-21 cells byelectroporation. The transfected cells were seeded in T-25 flasks andincubated for 24-48 h at 37 C and 5% CO₂. Virus was serially passed upto 4 times in BHK-21. Virus stocks at passages 4 and 5 in BHK-21 cellswere used in animal experiments and for the production of inactivatedvaccine and stored at −70° C. Virus titers were determined by plaqueassays as described below.

Example 6 Rescue of Parental and Mutant Viruses, Viral Growth, andPlaque Assays; In Vitro Characterization of Double Negative MarkerChimeric FMDV

BHK-21 cells were mock- or infected with the parent recombinant virusA₂₄WT, or chimeric viruses, A₂₄LL Asia1 3B_(PVKV)3D_(YR) and A₂₄LLTurkey 3B_(PVKV)3D_(YR) at a MOI of 5 pfu/cell. At 5 h pi, cell lysateswere collected and stored at −70° C. until further use. The doublenegative marker chimeric FMDV viruses A₂₄LL Asia1 3B_(PVKV)3D_(YR) andA₂₄LL Turkey 3B_(PVKV)3D_(YR), rescued from BHK-21-transfected cells,confirmed presence of Asia1 and Turkey capsid, respectively by sequenceanalysis. Positive Asia1-A₂₄LL3B_(PVKV)3D_(YR) clones were screened andidentified by PCR using Asia P1-specific sense primer P1679(5′-GCTGCCCTCGAAAGAGGGAATAG; SEQ ID NO:28) and A₂₄ specific antisenseprimer R10 (5′-AAACTTTTCTTCTGAGGCTATCCAT; SEQ ID NO:29) while positiveA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) were identified by PCR using A₂₄specific primer, L3 (5′-AGCACAGTAGCTTTGTTGTGAAACT; SEQ ID NO:30) andTurkey06 P1 specific primer 1615 (5′-CGCGCCGCAAGAGGCCCCAGGT; SEQ IDNO:31).

In addition, the presence of the chimera viruses in infected cellculture cells can be easily detected on an agarose gel following RNAextractions and RT-PCR reactions. Viral RNA of A₂₄LLAsia1-A₂₄LL3B_(PVKV)3D_(YR) and A/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) wereextracted from passage 4 using the RNeasy® Mini Kit (Qiagen) accordingto the manufacturer's instructions. One-Step Reverse TranscriptasePolymerase Chain Reaction (RT-PCR) (Invitrogen) was performed to detectthe presence and/or absence of specific mutations in the chimera FMDVviruses. Primers P1457 (5′-TGACTTCCA CGCAGGCATTTTCC; SEQ ID NO:32) andR8 (5′-TAGTTAAATGAAGCAGGAAGC TGT; SEQ ID NO:33) were used to detect theabsence of the leader gene while primers L5(5′-ACAACTACTACATGCAGCAATACCA; SEQ ID NO:34) and R6 (5′-AGTGAATTTGGAGTTTAGTCCAGTG; SEQ ID NO:35) were used to show the absence of the A₂₄capsid in the chimera viruses. Likewise, primers P1690(5′-GCCTGGCAAGTTCTGC ATTCAGTGG; SEQ ID NO:23) and P1634(5′-TCTCCGGCTAGCTTAAGCAGGTCAAA ATTCAGAAGCTGCTTCTCAGGTGCAATGA; SEQ IDNO:22) were used to detect Asia1 capsid while P1590(5′-GCTCCACTGACACTACCTCCAC; SEQ ID NO:36) and P1612(5′-GCCGGCGCTGACCGACACGACC; SEQ ID NO:37) were used to detect Turkey06capsid. In addition, primers L13 (5′-TTTTCAAACAGATCTCAATTCCTTC; SEQ IDNO:38) and R15 (5′-GCAAGCAAACTTGTATTCTCTTTTC; SEQ ID NO:39) were used todetect the two copies of 3B in the chimera viruses.

All positive clones were sequenced to verify the absence of the leadergene and presence of Asia1 or Turkey P1, 3B_(PVKV) and 3D_(YR)mutations. Complete viral sequences detect no other mutations except forthe expected lack of the leader coding region, modified 3B region andmutation at the 3D^(pol) locus encoding the YR epitope. FIG. 8B showsRT-PCR results (using A₂₄ capsid specific primers for L^(pro)) fail todetect that L^(pro) on A₂₄LL Asia1-A₂₄LL3B_(PVKV)3D_(YR) and A₂₄LLA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) infected cells extracts, indicatingthe lack of the two regions on these chimera viruses. As expected RT-PCRreactions using specific primers of Asia1 capsid sequences gave aproduct for Asia1-A₂₄LL3B_(PVKV)3D_(YR) virus while primers specific forTurkey06 capsid detected its presence onA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) but not on A₂₄WT virus. In addition,the presence of only two copies of 3B peptide-coding sequences can bedetected on the chimeric viruses compared to the A₂₄WT virus.

Plaque assays of Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) viruses show similar viral titers whencompared to the parental A₂₄WT virus (FIG. 8A). However, the plaquemorphology of the chimera viruses, as expected, is significantly smallerand therefore, requires a 72 h overlay incubation when compared to a 48h incubation of the A₂₄WT virus. In addition, in vitro growth kineticsof Asia1-A₂₄LL3B_(PVKV)3D_(YR) and A/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR)mutants relative to parental A₂₄WT virus were determined using a highmultiplicity of infection (MOI of 5) in BHK-21, LFBK cells and IBRS-2cells (FIG. 9). Titers of the parental A₂₄WT as well as the chimeraviruses, Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) peaked at 4 hours post infection (hpi)and remained the same over 30 h period in BHK-21 cells. However, thechimera viruses showed significantly restricted growth (˜2-3 logs lowertiters) in both bovine and swine cells when compared to the A₂₄WT virus.

For Western blot analysis, cell lysates were resuspended in STE buffer(10 mM Tris ph 8, 1 mM EDTA, 0.1M NaCl) supplemented with 1% Triton-X100and Benzonase (Novagen). 8 μl of cell lysates were run under denaturingconditions in a 12% SDS-PAGE gel (Invitrogen) and transferred ontonitrocellulose membranes using XCell II™ system (Invitrogen). The blotswere blocked with 5% skim milk in PBS-Tween (PBST) for 1 h at roomtemperature followed by an additional incubation of 1 h withFMDV-specific monoclonal antibodies (mAb F32-44 (1:5), mAb F19-6(1:100), mAb F8B (1:500) in 1% skim milk and PBST. Blots were thenwashed twice with PBST for 5 minutes each and incubated for anadditional hour with goat anti-mouse IgG antibody conjugated to horseradish peroxide (HRP) (1:20,000) diluted in 1% skim milk and PBST. Theblots were subsequently washed three times with PBST and developed withSuperSignal West Dura Extended Duration Substrate (ThermoScientific).Mouse anti-tubulin IgG conjugated to HRP was used at a dilution of 1:500as a loading control.

The antigenic profiles of the parental as well as chimera viruses wereexamined by western blots using MAbs F19-6, F32-44 and F8B. FIG. 10shows that BHK-21 cell lysates infected with the A₂₄WT virus reactedwith all three MAbs whereas both Asia1-A₂₄LL3B_(PVKV)3D_(YR) andA/Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) viruses failed to react with MAbs F8Band F32-44. These results indicate that mutations on the 3B region aswell as the 3D^(pol) epitope (two amino acids replacement) allowsdifferentiation of the chimeric viruses from wild-type virus by theirlack of reactivity with MAbs F8B and F32-44.

Example 7 Pathogenicity of A₂₄WT3D_(YR) and A₂₄LL3D_(YR) Viruses inCattle and Swine

To examine the influence of mutations on pathogenicity and theserological response to recombinant A₂₄WT3D_(YR) and A₂₄LL3D_(YR)mutants in comparison with the parental virus A₂₄WT in cattle, weperformed aerosol inoculation of these viruses in cattle, followed bymeasurement of clinical disease and virus shed to the environment.

Two Holstein steers for A₂₄WT and three for each mutant virus weremarked and housed in a single room. Prior to infection the animals weremoved to separate rooms and each of them were inoculated by aerosol witheither 1×10⁷ (for A₂₄WT) or 1-3×10⁶ TCID₅₀ (for A₂₄WT3D_(YR) andA₂₄LL3D_(YR) mutants) using a method previously described (Pacheco etal. 2010. Vet. J. 183:46-53). Sera and oral secretions were collecteddaily for up to 9 days for A₂₄WT and 21 days for A₂₄WT3D_(YR) andA₂₄LL3D_(YR) mutants, as well as temperature and clinical evaluation forthe same period of time. Shedding of virus in the air was also monitoredusing a Dry Filter Unit (DFU) Model 1000 air pump developed by theProgram Executive Office for Chemical Biological Defense (PEO-CBD).Clinical signs were scored as 1 credit for each affected foot and onecredit for the affected head (vesicles in mouth, nostrils, tongue orlips). FMDV RNA was measured in sera, swabs and air samples by rRT-PCRas described below.

Following inoculation, several parameters including fever, clinicalscore, viremia, neutralizing antibodies and the presence of virus in airand oral swabs samples were recorded and analyzed (Table 1). Nine steersallocated into independent rooms were aerosol-inoculated withapproximately 1×10⁷ TCID₅₀ of A₂₄WT or 1-3×10⁶ TCID₅₀ total of eitherA₂₄WT3D_(YR) or A₂₄LL3D_(YR) viruses. Animals inoculated with A₂₄WTvirus (bovines #7109, 7110) showed viremia, virus in saliva and fever by2 dpi. Clinical signs appeared by 2-4 dpi and reaching a high clinicalscore by 5-7 dpi when neutralizing antibodies were first detectable.Bovines #7199, 1 and 2 inoculated with A₂₄WT3D_(YR) virus showed viremiaby 2 dpi reaching a peak at 3-4 dpi. Virus was detectable in salivastarting at 2-3 dpi and reaching a peaking at 2-3 dpi. Fever appeared at2 dpi and lasted up to 3 or 5 dpi. Clinical signs appeared by 4-6 dpiand virus shed in the air started by 3 dpi. Serum neutralizingantibodies were first detectable by 5-6 dpi in all three cows (Table 1).Vesicular fluid was collected from lesions on the 4 and 6 dpi (bovine#1, 2 and 7199) and each sample was separately processed for RT/PCR andsequencing. These fluids contained viruses that were indistinguishablefrom the inoculated virus in their genome sequences, further indicatingthat the A₂₄WT3D_(YR) virus has not changed during growth in bovines. Inclear contrast with the pathogenic profile of A₂₄WT3D_(YR) and A₂₄WTviruses, the three animals inoculated with A₂₄LL3D_(YR) (bovine #7201, 3and 4; Table 1) showed absence of fever, viremia, clinicalmanifestation, or shedding of virus in saliva or air samples. The levelof attenuation was such, that these animals did not develop significantlevels of neutralizing antibodies during the course of the experiment(Table 1), even though antibodies against viral structural proteins weredemonstrated by 21 dpi by radioimmunoprecipitation assays (data notshown).

TABLE 1 Responses of Cattle to Infection with A₂₄WT, A₂₄WT3D_(YR) orA₂₄LL3D_(YR) Viruses Max Clinical Shedding Virus in Score/MaxNeutralization In Air Viremia Max Saliva Max Fever Achievable^(f)TiterMax^(h) TiterMax^(j) Bovine #^(a) Virus Titer^(b) (DPI)^(c)Titer^(b) (DPI)^(c) (DPI)^(c) (DPI)^(g) (Starting DPI)^(i) (DPI)^(c)7109 A₂₄WT 7.60 (3)  8.90 (3) Yes (2; 3) 5/5 (7)   2.4 (5) 5.57 (5) 7110A₂₄WT 7.31 (4) 10.18 (2) Yes (2 to 5) 5/5 (5)   2.4 (6) ND 7199A₂₄WT3D_(YR) 6.73 (3)  9.58 (6) Yes (6; 8) 1/5 (6)   2.7 (6) ND 1A₂₄WT3D_(YR) 7.55 (3)  8.00 (4) Yes (2; 3) 5/5 (6)   3.0 (5) 3.66 (7) 2A₂₄WT3D_(YR) 7.95 (3)  9.47 (4) Yes (2; 3) 2/5 (7)   3.0 (5) 2.55 (4)7201 A₂₄LL3D_(YR) Neg^(k) Neg No 0/5 <0.9 Neg^(l) 3 A₂₄LL3D_(YR) Neg NegNo 0/5 <0.9 Neg 4 A₂₄LL3D_(YR) Neg Neg No 0/5 <0.9 Neg ^(a)Bovines wereinoculated with 1× 10⁷ TCID₅₀ of A₂₄WT or 1-3 × 10⁶ TCID₅₀ of A₂₄WT[YR]and A₂₄LL[YR] of virus by aerosolization. ^(b)Log 10 RNA copy number/ml.^(c)Indicates the day after inoculation that peak of virus was detected.^(d)Fever defined as rectal temperature ≧40.0° C. ^(e)Indicates daysfever was detected. ^(f)Clinical scores were based on the number of feetwith vesicular lesions and lesions in the head (mouth, nostrils, lips ortongue), with a maximum of five. ^(g)Indicates the day after inoculationthat maximum score was reached. ^(h)FMDV-specific neutralizing antibodytiter (log 10 of reciprocal of the last serum dilution to neutralize 100TCID₅₀ of virus in 50% of the wells. ^(i)Indicates the first day afterinoculation that neutralizing antibodies were detected. ^(j)Log 10 RNAcopy number/1000 liters. ^(k)RNA copy number minimum detection level =10^(2.4)/ml. ^(l)RNA copy number minimum detection level = 10^(0.8)/1000liters. ND: Not Determined

Mutant (A₂₄LL3D_(YR)) virus was tested for its virulence in swine.Briefly, two 20-kg pigs were inoculated intradermally with 10⁵TCID₅₀ ofeach of the virus and 48 hours later two naïve pigs were added in directcontact. Sera, nasal and oral secretions were collected daily for up to21 days post inoculation (dpi), as well as rectal temperature andclinical evaluation. FMDV RNA was measured in sera, swabs and airsamples by rRT-PCR as described below.

Experimental inoculation in the heel-bulb of susceptible pigs with 10⁵TCID₅₀ of A₂₄LL[YR] virus was performed in two animals (Table 2, animals#40 and #41), and two contact animals (#43 and #44) were moved to theroom 48 hpi and housed together for 19 days. Among the inoculatedanimals, only one of the directly inoculated pigs (#41) had for one day(1 dpi) detectable RNA in serum (10^(5.44) viral RNA copy numbers perml) and 10^(5.45) viral RNA copy numbers per ml in oral swabs at 2 dpi.This animal also developed low serum neutralizing antibodies titersstarting at 4 dpi, but in the absence of any clinical manifestation ofFMD. Interestingly, no virus was shed from this pig to the seconddirectly inoculated pig, nor it did to the two in-contact animals (#43and #44). All pigs were never pirexic (temperatures remained below 40°C.) during the course of this experiment. Furthermore, none of thecontact pigs showed clinical signs of FMD or vesicular lesionsthroughout the experiment.

TABLE 2 Response of Swine: Direct Inoculation (Pigs #40, #41) or ContactInoculation (Pigs #42; #43) with 10⁵ TCID₅₀ of A₂₄LL3D_(YR.) Pig #40 0dpi^(a) 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi 6 dpi Viremia^(b)   Neg^(c) NegNeg Neg Neg Neg Neg Virus in Oral Swab^(b) Neg Neg Neg Neg Neg Neg NegVirus in Nasal Swab^(b) Neg Neg Neg Neg Neg Neg Neg NeutralizationTiter^(d) <0.9 <0.9 <0.9 <0.9 <0.9 <0.9 <0.9 Clinical Score Neg Neg NegNeg Neg Neg Neg Pig #41 0 dpi 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi 6 dpiViremia Neg 5.44 Neg Neg Neg Neg Neg Virus in Oral Swab Neg Neg 5.45 NegNeg Neg Neg Virus in Nasal Swab Neg Neg Neg Neg Neg Neg NegNeutralization Titer <0.9 <0.9 <0.9 <0.9 <0.9 1.2 1.2 Clinical Score NegNeg Neg Neg Neg Neg Neg Pig #42 0 dpc^(e) 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi6 dpi Viremia Neg Neg Neg Neg Neg Neg Neg Virus in Oral Swab Neg Neg NegNeg Neg Neg Neg Virus in Nasal Swab Neg Neg Neg Neg Neg Neg NegNeutralization Titer <0.9 <0.9 <0.9 <0.9 <0.9 <0.9 <0.9 Clinical ScoreNeg Neg Neg Neg Neg Neg Neg Pig #43 0 dpc 1 dpi 2 dpi 3 dpi 4 dpi 5 dpi6 dpi Viremia Neg Neg Neg Neg Neg Neg Neg Virus in Oral Swab Neg Neg NegNeg Neg Neg Neg Virus in Nasal Swab Neg Neg Neg Neg Neg Neg NegNeutralization Titer Neg Neg Neg Neg Neg Neg Neg Clinical Score Neg NegNeg Neg Neg Neg Neg ^(a)Days Post-inoculation. ^(b)Log 10 RNA copynumber/ml. ^(c)RNA copy number minimum detection level = 10^(2.4)/ml.^(d)FMDV-specific neutralizing antibody titer (log 10 of reciprocal ofthe last serum dilution to neutralize 100 TCID₅₀ of virus in 50% of thewells). ^(e)Days post-contact inoculation

Example 8 Double FMDV Negative Marker A₂₄LL3B_(PVKV)3D_(YR): Attenuatedin Cattle and Induction of an Immune Response Against the Non-StructuralViral Protein 3B

To examine the effect of epitope mutations on FMDV virulence in cattle,A₂₄WT3B_(PVKV) 3D_(YR) and A₂₄LL3B_(PVKV)3D_(YR) viruses were comparedin two groups of bovines inoculated with 10⁶ TCID₅₀ of virus andmonitored for clinical FMD. Results from this experiment are shown inTable 3. While A₂₄WT3B_(PVKV) 3D_(YR) was highly pathogenic, effectivelyinducing fever and clinical signs in cattle, double epitope mutantA₂₄LL3B_(PVKV) 3D_(YR) virus failed to induce FMD, neither producedvesicles at the site of inoculation. Attenuation of A₂₄LL3B_(PVKV)3D_(YR) virus was also reflected in lack of viremia or virus shedding(determined on bovine #9146) consistent with the in vivo results of thesingle marker A₂₄LL3D_(YR) mutant virus (see Tables 1 and 2).Conservation of 3B and 3D^(pol) epitope mutations were confirmed bynucleotide sequence analysis of virus from tissues or vesicular fluidrecovered from A₂₄WT3B_(PVKV) 3D_(YR)-infected animals (data not shown).Antibody responses to FMDV 3B epitopes were measured by an in housecELISA using MAb F8B as competitor and a 3B peptide (see Material andMethods). Sera collected from animals inoculated with the markerA₂₄LL3B_(PVKV)3D_(YR) virus by the aerosol route (days 0 and 21) orinoculated with A₂₄LL3B_(PVKV) 3D_(YR) by the intradermolingual (days 0and 21) route were analyzed against FMDV 3B peptide in a cELISA (FIG.7). Results showed that the sera from A₂₄WT3B_(PVKV)3D_(YR) (bovine#9143 and 9144) or A₂₄LL3B_(PVKV) 3D_(YR) (bovine #9145 and 9146) doublemutant-infected animals at day 21 post-infection were unable to competewith MAbF8B as the sera obtained from these animals prior to infection(day 0, uninfected animals) were, indicating a lack of anti-3B epitopeantibodies (FIG. 7). The presence of neutralizing antibody responsemeasured at day 14 (Table 3) showed titers of 1.5 and 2.5 for bovines#9145 and #9156 (infected with LL double mutant) and of 3.6 and 3.0 forthe WT double mutant-infected animals #9143 and #9144.

TABLE 3 Virus titers, clinical samples and virus shedding measurementsfrom cattle following infection with A₂₄WT3B_(PVKV)3D_(YR) orA₂₄LL3B_(PVKV)3D_(YR) viruses. Virus in Maximum Viremia, Saliva,Clinical Score/ Shedding in Maximum Maximum Maximum air. MaximunTiter.^(b) Titer^(b) Fever^(d) achievable^(f) Neutralization Titer^(i)Bovine #^(a) Virus (DPI)^(c) (DPI)^(c) (DPI)^(e) (DPI)^(g) Titer at 14DPI^(h) (DPI)^(c) 9143 A₂₄WT3B_(PVKV)3D_(YR) 7.40 (4) 9.03 (5) Yes (3)1/5 (5) 3.6 6.29 (6) 9144 A₂₄WT3B_(PVKV)3D_(YR) 7.92 (4) 8.85 (5) Yes(4) 4/5 (7) 3.0 5.45 (7) 9145 A₂₄LL3B_(PVKV)3D_(YR) Negative Negative No0/5 1.5 ND^(j) 9146 A₂₄LL3B_(PVKV)3D_(YR) Negative Negative No 0/5 2.4Negative ^(a)Bovines were inoculated with 7 × 10{circumflex over ( )}6TCID₅₀ of A₂₄WT3B_(PVKV)D_(YR) by aerosolation or with 1 × 10{circumflexover ( )}6 TCID₅₀ of A₂₄LL3B_(PVKV)3D_(YR) virus by theintradermolingual route. ^(b)Log10 RNA copy number/ml. ^(c)Indicates theday after inoculation that peak of virus was detected ^(d)Fever definedas rectal temperature ≧40.0° C. ^(e)Indicates days fever was detected.^(f)Clinical scores were based on the number of feet with vesicularlesions and lesions in the head (mouth, nostrils, lips or tongue), witha maximum of five. ^(g)indicates the day after inoculation that maximumscore was reached. ^(h)FMDV-specific neutralizing antibody titer (log10of reciprocal of the last serum dilution to neutralize 100 TCID₅₀ ofvirus in 50% of the wells) ^(i)Log10 RNA copy number/1000 liters ^(j)ND:not determined

Example 9 Antigen Production and Vaccine Formulation

The A₂₄LL3D_(YR) vaccine antigen was harvested from infected BHK-21monolayers cells and inactivated with 5 mM BEI for 24 h at 25° C. Theinactivated antigen was then concentrated and partially purified with 8%polyethylene glycol 8000. The vaccine was prepared aswater-in-oil-in-water (WOW) emulsion with Montadine ISA 206 (Seppic,Paris) according to the manufacturer instructions. Briefly, the oiladjuvant was mixed into the aqueous antigen phase (50:50) at 30° C. for15 minutes and stored at 4° C. for 24 hours, followed by another briefmixing cycle for 10 minutes. The integrity of 146S particles and antigenconcentration present in the experimental vaccine was determined by10-30% sucrose density gradient and 260 nm densitometry.

The commercial vaccine used for comparison was a polyvalent vaccine(Biogenesis-Bagó Bioaftogen serie 565 composed of O₁ Campos, A₂₄Cruzeiro, A Arg 2001 and C₃ Indaial.

Example 10 Vaccination with BEI-Inactivated A₂₄LL3D_(YR) Virus ProtectsCattle Against Challenge with Pathogenic A₂₄WT FMDV

To determine the efficacy of the marker vaccine in providingimmunological protection against challenge with parental A₂₄WT virus,the BEI-inactivated A₂₄LL3D_(YR) vaccine was tested in parallel with acommercial FMDV vaccine in a cattle vaccine trial. Ten Holstein steers,between 250 and 300 kg, were allowed to acclimatize from shipping for 1week before testing was initiated. Eight steers were vaccinated witheither the commercial vaccine (cattle 863, 864, 865 and 866) or withA₂₄LL3D_(YR)/water-in-oil-in-water (WOW, cattle 867, 868, 869, 870)vaccine, intramuscularly in the neck. As shown in Table 4, four cattle(#863-866) were each inoculated intramuscularly with 1 dose of acommercial trivalent vaccine and four other steers (#867-870) receivedthe A₂₄LL3D_(YR) BEI-inactivated vaccine. Cattle 871 and 872 werevaccinated with sterile PBS to be used as unvaccinated controls.

TABLE 4 Specific neutralizing antibody response against FMDV A₂₄WT aftervaccination with commercial polyvalent or A₂₄LL3D_(YR) vaccine andchallenged with FMDV A₂₄WT. Bovine Days Post Vaccination # 0^(a) 7 1421^(b) 28 35 42 Commercial 863 <0.9^(c) 0.9 1.2 1.5 3.9 4.2 3.9 Vaccine864 <0.9 1.2 0.9 0.9 3.6 3.6 3.6 865 <0.9 0.9 0.9 1.8 3.9 3.6 3.9 866<0.9 1.2 1.5 2.1 ND ND ND 867 <0.9 2.1 2.4 2.7 3.3 3.3 3.0 A₂₄LL3D_(YR)868 <0.9 2.4 2.1 2.7 3.3 3.6 3.9 Vaccine 869 <0.9 2.1 2.4 2.7 3.0 2.73.0 870 <0.9 2.1 1.8 2.4 3.0 3.3 3.0 PBS 871 <0.9 <0.9 <0.9 <0.9 2.4 3.03.6 (Controls) 872 <0.9 <0.9 <0.9 <0.9 2.7 3.3 3.6 ^(a)Day ofVaccination ^(b)Day of Challenge ^(c)Virus neutralizing titers of serumantibodies

On day 21 post vaccination all 10 cattle were challengedintradermolingually with 10⁴ BTID₅₀ (50% bovine tongue infectious doses;In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of2009, edited and published by OIE—The World Organization for AnimalHealth, Foot and mouth disease, Chapter 2.1.5) of parental A₂₄WT. Theanimals were then monitored at 0, 4, 7 and 10 days post-challenge forthe appearance of localized and generalized lesions and rectaltemperatures were recorded. Sera, nasal swabs (cotton tip, immersed in 2ml of minimum essential medium with 25 mM HEPES and 1% FBS) andtemperature were collected daily. Clinical signs were scored as 1 creditfor each affected foot, presence of vesicles in the head was notconsidered due to lingual inoculation of challenge. FMDV RNA wasmeasured in sera, swabs and air samples by rRT-PCR as described below.

The 8 immunized cattle, regardless of the vaccine they received, wereprotected from challenge with parental A₂₄WTvirus as observed by theabsence of generalized vesicles or high temperatures. All vaccinatedanimals developed a detectable FMDV-specific neutralizing antibodyresponse by 7 days post-vaccination (dpv), with slightly higherneutralizing titers for our experimental A₂₄LL3D_(YR) vaccine morelikely due to a higher content of antigen mass (approximately 23 ug/doseA₂₄LL3D_(YR)/dose). By day 21 all animals but one (#864) had increasedtiters of serum neutralizing antibodies. In contrast, the un-vaccinatednaïve animals (bovines #871, 872) that received PBS had no detectableFMDV-specific antibody response (Table 4).

Twenty one days dpv, all animals were challenged by intradermalinoculation at four sites in the tongue with 10,000 bovine infectiousdoses (BTID₅₀) of parental FMDV A₂₄WT. Both control animals developedfever within 24-72 h post challenge (dpc) while only one of thecommercial vaccine-immunized animals developed fever at 3 and 4 dpc. Allother animals showed no fever during the experiment (Table 5). Bothnaïve animals developed lesions on all four feet. In contrast, none ofthe vaccinated animals showed signs of FMD during the term of thisexperiment (up to 10 dpc, Table 6). Both control animals showed viremiaat 1 to 5 dpc and no virus was detected in the sera of any of thevaccinated animals (data not shown). Virus was detected from nasalsecretions in all animals, either vaccinated or not (data not shown).

TABLE 5 Rectal Temperatures^(b) of cattle (# 863-872) after challengewith FMDV A₂₄WT. Days Post Vaccination # 21^(a) 22 23 24 25 26 27 28 2930 31 32 Commercial 863 38.8 39.1 39.6 40.8 40.3 39.3 39.7 39.6 39.239.1 39.1 39.1 Vaccine 864 39.0 38.9 39.1 39.3 39.3 39.6 39.3 39.2 39.339.2 39.1 38.9 865 38.9 39.4 39.7 39.9 38.9 39.4 39.8 39.4 39.6 39.239.3 39.0 A₂₄LL3D_(YR) 867 39.1 39.9 39.8 39.9 39.7 39.8 39.7 39.4 39.339.6 39.1 39.0 Vaccine 868 38.7 39.7 38.9 39.0 38.9 39.3 39.2 39.3 39.439.2 39.3 38.9 869 39.1 39.7 39.9 39.7 39.1 39.4 39.4 39.7 39.8 39.439.1 39.2 870 39.0 39.1 38.8 39.2 39.1 39.3 39.4 39.0 39.1 39.2 39.238.9 PBS 871 38.6 38.9 40.1 40.7 39.3 39.3 39.1 39.1 39.2 39.3 38.9 39.0(Controls) 872 39.1 41.1 41.2 40.6 39.9 39.9 39.4 39.3 39.8 39.0 39.139.2 ^(a)Day of challenge ^(b)Temperature (° C.); Considered fever when≧40.0° C.

TABLE 6 Assessment of Clinical scores ^(a) of cattle after challengewith FMDV A₂₄WT^(b). Days Post Vaccination Bovine # 21^(c) 25 28 32Commercial 863 0 0 0 0 Vaccine 864 0 0 0 0 865 0 0 0 0 A₂₄LL3D_(YR) 8670 0 0 0 Vaccine 868 0 0 0 0 869 0 0 0 0 870 0 0 0 0 PBS 871 0 4 4 4(Controls) 872 0 4 4 4 ^(a) Clinical scores were based on the number offeet with vesicular lesions. ^(b)Cattle were challenged byintradermolingual inoculation of 4 log 10 bovine infectious doses ofFMDV A₂₄WT. ^(c)Day of challenge

Example 11 Pathogenic Characteristics of Double Marker A-Turkey/06/andAsia1/LL3B_(PVKV)3D_(YR) Recombinant Viruses in Pigs

Experimental direct inoculation in the heel-bulb of susceptible pigswith 10⁶ PFUs of Asia1-A₂₄LL3B_(PVKV)3D_(YR) virus was performed inthree animals (Table 7, animals #199, 200 and #201), and two contactanimals (#197 and #198) that were moved into the same pen at 24 hpi andhoused together for 20 days. Likewise, pigs #202, 203 and 204 (Table 8)were inoculated in a separate room with 10⁶ PFUs ofA-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) virus and pigs #205 and 206 were movedinto the room as in contact control animals. No clinical signs of FMDwere observed in any of the directly inoculated or contact animalsduring the course of this experiment. Among theAsia1/A₂₄LL3B_(PVKV)3D_(YR) directly inoculated animals, one of threeanimals had traces of detectable RNA in serum at 2 dpi and developedantibodies against FMDV at days 7 (Pig 199 neutralizing titer of 1.5).Among the A-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR) direct inoculated pigs, onepig (#204) had traces of serum and oral swab at 7 dpi but did notdeveloped FMDV-specific antibodies. However, another pig (#202) showedno virus in serum or swabs but had a serum neutralizing titer of 1.8 at14 dpi, in the absence of any clinical manifestation of FMD.Interestingly, no virus was shed from the three inoculated animals tothe two in-contact animals in either group. The pigs were never pirexic(temperatures remained below 40° C.) during the course of thisexperiment. Furthermore, in postmortem examination of various tissuesfrom two animals in each of the inoculated groups (Table 8), only twopigs inoculated with A-Turkey/06-A₂₄LL3B_(PVKV)3D_(YR), presented twoFMDV-RNA positive tissues, one in each pig, with very low amounts of RNAcopy numbers. Shedding of virus was also measured through air samplingand traces of RNA was detected at 3 and 4 dpi in the air filters locatedin the room that contained the pigs inoculated withAsia1-A₂₄LL3B_(PVKV)3D_(YR).

TABLE 7 Responses of swine to infection with Asia1-A₂₄LL3B_(PVKV)3D_(YR)or ATurkey06-A₂₄LL3B_(PVKV)3D_(YR) viruses; virus shedding measurements.Viremia Virus in Saliva - Virus in Nasal Swine Inoculation Max Titer^(a)Max Titer^(a) Swab - Max Fever^(c) Clinical Neutralization # Virus Route(DPI)^(b) (DPI)^(b) Titer^(a) (DPI)^(b) (DPI) Score Titer^(d) (DPI)^(e)199 Asia1/A₂₄LL3B_(PVKV)3D_(YR) Direct^(f) 4.03 (2)^(h) NegativeNegative No Negative 1.5 (7)  200 Asia1/A₂₄LL3B_(PVKV)3D_(YR) DirectNegative Negative Negative No Negative <0.9 201Asia1/A₂₄LL3B_(PVKV)3D_(YR) Direct Negative Negative Negative NoNegative <0.9 197 Asia1/A₂₄LL3B_(PVKV)3D_(YR) Contact^(g) NegativeNegative Negative No Negative <0.9 198 Asia1/A₂₄LL3B_(PVKV)3D_(YR)Contact Negative Negative Negative No Negative <0.9 202ATurkey06/A₂₄LL3B_(PVKV)3D_(YR) Direct Negative Negative Negative NoNegative 1.8 (14) 203 ATurkey06/A₂₄LL3B_(PVKV)3D_(YR) Direct NegativeNegative Negative No Negative <0.9 204 ATurkey06/A₂₄LL3B_(PVKV)3D_(YR)Direct 3.34 (1) 3.40 (7) Negative No Negative <0.9 205ATurkey06/A₂₄LL3B_(PVKV)3D_(YR) Contact Negative Negative Negative NoNegative <0.9 206 ATurkey06/A₂₄LL3B_(PVKV)3D_(YR) Contact NegativeNegative Negative No Negative <0.9 ^(a)Expressed in log10 RNA copynumber/ml. ^(b)Indicates the day after inoculation that peak of viruswas detected ^(c)Fever defined as rectal temperature ≧40.0° C.^(d)FMDV-specific neutralizing antibody titer (log10 of reciprocal ofthe last serum dilution to neutralize 100 TCID50 of virus in 50% of thewells). ^(e)Indicates first day(s) after inoculation that neutralizingantibodies were detected. ^(f)Indicates intradermal inoculation in theheel bulb with 10{circumflex over ( )}6 TCID50 ^(g)Direct contactstarted 24 hours post direct inoculation and lasted until the end of theexperiment (21 dpi). ^(h)RNA copy number, sensitivity = 10{circumflexover ( )}2.4/ml

TABLE 8 Real time measurements of viral RNA on postmortem samples fromswine inoculated with Asia1-A₂₄- LL3B_(PVKV)3D_(YR) orATurkey06-A₂₄LL3B_(PVKV)3D_(YR). Asial-A₂₄- ATurkev06-A₂₄-LL3B_(PVKV)3D_(YR) LL3B_(PVKV)3D_(YR) Animal # Animal # Tissue 199 200203 204 Inoculation site NEG ^(a) NEG NEG NEG Tongue NEG NEG NEG NEGPopliteal LN NEG NEG 2.36 NEG Nasopharynx NEG NEG NEG 2.34 Lung NEG NEGNEG NEG Palatine tonsil NEG NEG NEG NEG Coronary band NEG NEG NEG NEG^(a) Indicates FMDV RNA copy number per mg of tissue. Cutoff value is2.26.

Example 12 BEI-Inactivated A₂₄LL3B_(PVKV)3D_(YR) and Asia1-LL3B_(PVKY)3D_(YR) Viruses Elicit Protective Immune Responses inCattle

To determine the efficacy of the marker BEI-inactivatedA₂₄LL3B_(PVKY)3D_(YR) and chimeric Asia 1-LL3B_(PVKY)3D_(YR) vaccines inproviding immunological protection against challenge with parental A₂₄and Asia-1 viruses, the BEI-inactivated A₂₄LL3B_(PVKY)3D_(YR) andchimeric Asia 1-LL3B_(PVKY)3D_(YR) vaccines were tested in a cattlevaccine trial. Vaccine antigen was harvested and inactivated as shown inExample 9. Four steers (Cattle #10-18, 10-19, 10-20 and 10-21) receiveda 15 μg/dose of BEI-inactivated A₂₄LL3B_(PVKY)3D_(YR) vaccine as shownin Table 9. As a control, steers #10-22 and #10-23 received a solutioncontaining PBS/adjuvant. Four steers (Cattle #11-10, 11-11, 11-12 and11-13) received a 9 μg/dose of BEI-inactivated Asia 1-LL3B_(PVKY)3D_(YR)vaccine; control animals #11-14 and 11-15 were treated as above.

TABLE 9 Vaccination: Clinical scores^(a) after challenge with FMDV A₂₄WTor Asia1. Days post vaccination Bovine # 21 ^((b)) 24 28 31A₂₄LL3B_(PVKV)3D_(YR) Vaccine 10-18 0 0 0 0 15 ug /dose 10-19 0 0 0 010-20 0 0 0 0 10-21 0 0 0 0 Asia1 A₂₄LL3B_(PVKV)3D_(YR) 11-10 0 3 4 4Vaccine 11-11 0 0 0 0 9 ug /dose 11-12 0 0 0 0 11-13 0 0 0 0 PBS(Controls) 10-22 0 4 4 4 10-23 0 4 4 4 11-14 0 4 4 4 11-15 0 4 4 4^(a)Clinical scores were based on the number of feet with vesicularlesions. ^((b))Day of challenge. Cattle were challenged byintradermolingual inoculation of 10,000 bovine infections doses ofhomologous FMDV.

On day 21 post vaccination all animals were challenged by intradermalinoculation at four sites in the tongue with 10,000 bovine infectiousdoses (BTID₅₀) of parental FMDV A₂₄Cru or Asia1 viruses. The animalswere then monitored at 0, 4, 7 and 10 days post-challenge for theappearance of localized and generalized lesions and rectal temperatureswere recorded.

All control mock-vaccinated animals developed lesions on their feet(Table 9) and viremia at 1 to 5 dpc (not shown) while only one of theAsia1-A₂₄LL3B_(PVKV)3D_(YR) vaccine-immunized animals (bovine #11-10)developed fever at 48 h dpc and showed vesicles characteristic of FMD inthree feet. As shown in Table 9, all vaccinated animals inoculated withthe double marker A₂₄LL3B_(PVKV)3D_(YR) vaccine and 3 of 4 animals thatreceived the Asia1-A₂₄LL3B_(PVKV)3D_(YR) inactivated vaccines were fullyprotected from challenge with the corresponding parental A₂₄WT or Asia 1viruses, as observed by the absence of clinical signs (Table 9) or hightemperatures (Table 10).

In the marker virus-vaccinated groups, animals developed detectableFMDV-specific neutralizing antibody responses by 7 or 14 dpv, withslightly higher neutralizing titers at 14 and 21 dpv except for theanimal #11-10 that maintained neutralizing titers at 1.5 at 14 and 21dpv (Table 11). All the control naïve animals (bovines #10-22, 10-23,11-14 and 11-15) that received PBS/adjuvant had no detectableFMDV-specific antibody response at day of challenge (Table 11). Viruswas detected from nasal secretions in all the animals post-challenge(data not shown).

TABLE 10 Vaccination Trial: Rectal temperatures (in ° C.) afterchallenge with parental FMDV A₂₄WT and Asia 1 Viruses. Days postvaccination Bovine # 21^(a) 22 23 24 25 26 27 28 29 30 31A₂₄LL3B_(PVKV)3D_(YR) Vaccine 10-18 38.8^(b) 38.8 38.7 39.0 39.0 39.238.9 39.0 39.4 38.7 39.2 10-19 38.3 38.8 38.8 39.0 38.6 39.4 38.7 38.439.0 38.9 38.7 10-20 38.4 38.8 38.2 38.7 39.0 39.7 39.1 38.7   ND^(c) NDND 10-21 38.6 38.4 38.8 38.9 38.4 38.8 38.8 38.7 38.8 38.4 38.9 Asia1A₂₄LL3B_(PVKV)3D_(YR) 11-10 38.4 39.7 41.0 40.7 40.0 39.5 38.4 38.2 38.838.2 38.6 Vaccine 11-11 38.7 38.8 40.3 39.1 38.6 38.6 38.9 38.8 39.238.8 38.8 11-12 38.3 38.8 39.0 38.7 38.3 38.6 38.6 38.8 38.9 38.7 38.411-13 38.1 38.7 39.3 39.3 38.7 38.8 38.7 38.6 38.8 38.6 38.6 PBS(Controls) 10-22 38.6 39.4 39.8 40.1 39.0 38.8 38.9 38.7 39.4 40.0 38.910-23 38.7 39.2 40.6 39.8 39.8 38.9 39.0 39.0 39.1 38.8 38.4 11-14 38.438.8 40.5 40.3 40.1 39.2 38.7 39.2 39.3 39.0 39.0 11-15 38.1 41.4 41.440.7 39.2 39.7 38.9 38.6 39.3 38.6 38.5 ^(a)Day of challenge bConsideredfever when ≧40.0° C. ^(c)Not determined

TABLE 11 Vaccination Trial: Specific neutralizing antibody responseagainst FMDV after vaccination with A₂₄LL3B_(PVKV)3D_(YR) orAsia1-A₂₄LL3B_(PVKV)3D_(YR) vaccines and challenged with FMDV A₂₄WT orAsia1. Days post vaccination Bovine # 0^(a) 7 14 21^(b) 28 35 42A₂₄LL3B_(PVKV)3D_(YR) 10-18   <0.9^(c) 1.8 2.1 2.1 3.0 3.3 3.0 Vaccine10-19 <0.9 2.1 2.1 2.1 3.3 3.6 3.0 15 ug/dose 10-20 <0.9 <0.9 2.1 1.82.7  ND^(d) ND 10-21 <0.9 1.5 1.8 2.1 2.1 2.7 3.6 Asia1 11-10 <0.9 1.21.5 1.5 ND ND ND A₂₄LL3B_(PVKV)3D_(YR) 11-11 <0.9 1.8 2.1 2.1 ND ND NDVaccine 11-12 <0.9 1.8 2.4 2.4 ND ND ND 9 ug/dose 11-13 <0.9 2.1 2.4 1.5ND ND ND PBS (Controls) 10-22 <0.9 <0.9 <0.9 <0.9 2.4 3.3 2.7 10-23 <0.9<0.9 <0.9 <0.9 2.7 2.7 2.1 11-14 <0.9 <0.9 <0.9 <0.9 ND ND ND 11-15 <0.9<0.9 <0.9 <0.9 ND ND ND ^(a)Day of vaccination. ^(b)Day of challenge^(c)Virus neutralizing titers of serum antibodies responses. ^(d)Notdetermined

Example 13 Foot and Mouth Disease Virus RNA Detection and DNA SequenceAnalysis

Fifty μl of each sample (sera or nasal swab resuspension) for each cowwere transferred to 96-well plates (King Fisher number 97002540)containing 150 μl lysis/binding solution. RNA was then extracted usingAmbion's MagMax-96 Viral RNA Isolation Kit (Ambion, catalogue number1836) on a King Fisher-96 Magnetic Particle Processor (Thermo ElectronCorp.). Briefly, after the initial 5 min lysis/binding step, the RNAsample underwent a series of four washing steps, a drying step, and afinal elution step. RNA was eluted in a final volume of 25 μl. At eachof the above steps, RNA was magnetically bound to the beads contained inthe lysis/binding solution and was transferred to the differentextraction solutions. For filters containing the air samples, ¼ filterswere processed with 600 μL RLT/β-mercaptoethanol and 106-micron acidwashed glass beads. The sample was then disrupted using a Retsch tissuelyser (model MM400) at 30 beats/sec for 3 minutes and the liquidsuspension used for RNA extraction with the standard RNeasy RNAextraction. RNA extracted from all the previous described samples wasanalyzed by rRT-PCR using 2.5 μl of RNA on the ABI 7000 as previouslydescribed (Callahan et al. 2002. J. Am. Vet. Med. Assoc. 220:1636-1642).The cutoff to consider a positive value for preclinical samples (seraand swabs) was 10^(2.4) RNA copy number/ml and for air samples, 10^(0.8)RNA copy number/1000 liters of air. When necessary, PCR amplicons weresequenced using gene-specific primers, Big Dye Termination CycleSequencing Kits (Applied Biosystems, Foster City, Calif.) and a PRISM3700 automated sequencer (Applied Biosystems). Primers and probes weredesigned using Primer Express® software (Applied Biosystems, FosterCity, Calif.).

Example 14 Monoclonal Antibodies, Expression of Recombinant FMDV-3DProtein

Sera taken at different times post infection were examined for thepresence of antibodies that could compete. Expression vectors for3D^(pol) were prepared by using standard recombinant DNA methods.Briefly, PCR was used to amplify the 3D^(pol)-coding sequence of a typeA FMDV. Forward primer P727 (5′-GCGGAATTCCCGCGGTGGA GGGTTAATCGTTGATAC;SEQ ID NO:17) designed to fuse the carboxi-terminal three amino acids ofubiquitin (Wei et al. 2001. J. Virol. 75:1211-1219.) to the codingsequence for 3D^(pol) were designed to include SacII restriction sitefor cloning purposes. The antisense primer encoded the carboxi-terminalresidues also contains a BamHI restriction site P728(5′-GCGGAATTCGGATCCTGCGTCACCGCACACGGCGTTCA CCC; SEQ ID NO:18). The PCRproduct was cloned into pET26cHis.

Example 15 Serology and Antigen Differentiation Assays

Serum samples from all animals from all trials were tested for thepresence of neutralizing antibodies against FMDV in a serumneutralization assay. Neutralizing titers were reported as thereciprocal of the last serum dilution to neutralize 100 TCID₅₀ ofhomologous FMDV in 50% of the wells (Golde et al. 2005. Vaccine23:5775-5782). Sera were also tested for the presence of antibodiesagainst viral proteins by a RIP assay as previously described (Picconeet al., supra).

To study the anti-3DPO′ response in the animals, we utilized seracollected from cattle inoculated by aerosol with either A₂₄WT3D_(YR)(bovines #s 7199, 1, 2), A₂₄WT (bovines #7109, 7110), or two A_(2A)WTintrodermolingual inoculated (#871, 872). Competitive Enzyme-LinkedImunosorbent Assay (cELISA) was performed following the protocol by Yanget al. (2007a, supra) with minor modifications. Briefly, recombinant3D^(pol) was diluted in buffer carbonate-bicarbonate (pH 9.6) to obtain0.33 μg/ml and 100 μl/well were used to coat Nunc Maxisorp plates(Fisher Scientific). Following 2 hours incubation at 37° C. on a rotaryshaker, plates were washed four times with 0.01 PBS, 0.05% Tween20(PBS-T), and triplicates of 50 μl/well of test sera (1/5 in PBS-T) and50 μl/well of F32-44 hybridoma culture supernatant (1/5 in PBS-T) wereapplied to the coated plates and incubated overnight at 4° C. Afterwashing four times, 100 μl/well of peroxidase-labeled goat antibody tomouse-IgG (H+L) (KPL) diluted 1/2000 in 5% skim milk in PBS-T were addedand incubated for 1 h at 37° C. After four washes the antigen-antibodiescomplexes were detected by the addition of 100 μl/well of SureBlueReserve™ (KPL) and stopped in 10 min with 50 μl/well of TMB BIueSTOP™solution (KPL). The OD was determined at 630 mm on an automated ELISAplate reader. For cELISA based on MAb F8B a similar protocol wasutilized with minor modification. In particular, the antigen applied tothe plate consisted of a peptide encoding the 3B sequence GPYAGPLETQKPLK(SEQ ID NO:40) applied at a concentration of 0.05 μg/well. Test serawere assayed at a 115 dilution in PBS-T and MAb F8B was used at a 1/125dilution.

Distinctive antibody responses were identified for mutant A₂₄WT3D_(YR)and A₂₄LL3D_(YR) against 3D^(pol) using a cELISA assay. Sera collectedfrom animals inoculated with the marker A₂₄WT3D_(YR) virus by theaerosol route (days 0 and 21) or inoculated with A₂₄WT by theintradermolingual (days 0 and 21) or the aerosol routes (days 0 and 9)were analyzed against FMDV 3D^(pol) protein in a cELISA. Arepresentative assay presented in FIG. 4 allows distinction of theserological responses in cattle to inoculation with either parentalvirus (A₂₄WT) and mutant (A₂₄WT3D_(YR)) virus. While seroconversionafter inoculation with A₂₄WT resulted in significant inhibition of theanti-3D^(pol) response in our cELISA format, sera from animalsinoculated with A₂₄WT3D_(YR) showed restricted inhibition. This assaythat utilizes monoclonal F32-44 allowed differentiation of animalsinfected with A₂₄WT from the negative marker virus A₂₄WT3D_(YR).

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

The foregoing description and certain representative embodiments anddetails of the invention have been presented for purposes ofillustration and description of the invention. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed. Itwill be apparent to practitioners skilled in this art that modificationsand variations may be made therein without departing from the scope ofthe invention.

We claim:
 1. A method of distinguishing between an animal exposed towild-type Foot and Mouth disease virus (FMDV) and an animal vaccinatedwith a marker FMDV vaccine, where the marker is a replacement of aminoacids His₂₇ and Asn₃₁ of FMDV 3D^(pol) with the amino acids Tyr and Argof the 3D^(pol) of Bovine Rhinovirus type 2 (BRV2), said methodcomprising: testing serum from an animal in a competitive enzyme-linkedimmunosorbent assay (cELISA) with monoclonal antibody (MAb) F32-44(Accession No. 130514-02), wherein inhibition of binding of MAb F32-44to the 3D^(pol) antigen of FMDV indicates an animal exposed to wild-typeFMDV, and lack of inhibition indicates an animal vaccinated with themarker FMDV vaccine.
 2. A method of distinguishing between an animalexposed to wild-type FMDV and an animal vaccinated with a double markerFMDV vaccine, where the double marker is the replacement of amino acidsHis₂₇ and Asn₃₁ of FMDV 3D^(pol) with the amino acids Tyr and Arg of the3D^(pol) of Bovine Rhinovirus type 2 (BRV2), and the replacement ofamino acids RQKP of FMDV 3B with the amino acids PVKV of the 3B of BRV2,said method comprising: testing serum from an animal in cELISA with MAbF8B (Accession No. 130514-01) and MAb F32-44 (Accession No. 130514-02),wherein inhibition of binding of MAb F8B to the 3B antigen of FMDV andinhibition of the binding of MAb F32-44 to the 3D^(pol) antigen of FMDVindicates an animal exposed to wild-type FMDV, and lack of inhibitionindicates an animal vaccinated with the double marker FMDV vaccine. 3.The method of claim 2, wherein the 3B antigen in the competitiveimmunoassay is the 3B peptide GPYAGPLETQKPLK (SEQ ID NO:40).
 4. Themethod of claim 1 where the serum is obtained from an animal suspectedof vaccination with a vaccine comprising a genetically modified FMDVencoded by the DNA of SEQ ID NO:1.
 5. The method of claim 2, where theserum is obtained from an animal suspected of vaccination with a vaccinecomprising a genetically modified FMDV encoded by the DNA of SEQ IDNO:3.
 6. The method of claim 1 or claim 2, where the serum is obtainedfrom an animal suspected of vaccination with a chimeric marker FMDVvaccine, where the capsid region of the marker FMDV has been replacedwith the capsid region of a different strain of FMDV.